Method for changing the intercellular mobility of an mRNA

ABSTRACT

The present invention relates to a method for changing the intercellular mobility of an min RNA of a gene in an organism, comprising: modifying a t RNA-like structure present in the m RNA by mutating the gene from which the m RNA is transcribed, or including the sequence of at RNA-like structure in the transcribed part of the gene. The method is in particular suited for plants. The intercellular mobility can be between different organs. Mutating the gene is for example for inducing loss of mobility of the transcript and comprises deleting the sequence of the tRNA-like structure from the gene, mutating the sequence of the t RNA-like structure to change the tridimensional configuration thereof, or inserting a genetic element into the gene to remove the tRNA-like structure from the transcribed part of the gene, or for inducing a change in the destination of the transcript and comprises modifying the sequence of the t RNA-like structure from the gene such that the transcript is addressed to a location different from the original destination of the transcript.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 filing of International PatentApplication No. PCT/EP2017/059029, filed Apr. 13, 2017, which claimspriority to International Patent Application No. PCT/EP2016/058282,filed Apr. 14, 2016, the entire contents of which are incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to a method for changing the intercellularmobility of an mRNA of a gene in an organism.

BACKGROUND

One of the most fundamental principles in biology is the relationshipbetween DNA, RNA and proteins. Generally, in eukaryotic organisms, DNAcomprises genes, which can be transcribed into messenger RNA (mRNA)molecules, and these mRNA molecules are subsequently translated intoproteins. These different steps take place at different locations in aeukaryotic cell: the DNA is contained inside an organelle which islocated inside the cytoplasm of a eukaryotic cell (i.e. a nucleus,mitochondrion or chloroplast). However, the mRNA transcript needs to beexported out of that organelle, because translation typically takesplace in the cytoplasm of the cell, which is where the ribosomesrequired for translation are located.

Thus, it is fundamental for the normal functioning of biological systemsthat mRNA transcripts are transported across organellar membranes, fromone subcellular compartment to another subcellular compartment. In thismanner, a cell's genetic information is efficiently translated into alarge number of different proteins that work together to enable the cellto maintain its own functionality and metabolism, and to respondadequately to its environment and to signals from other cells in theorganism, and to signals from the organism's environment. This so-calledcell-autonomy thus leads to a situation in which a cell's phenotype andbehaviour is directly linked to that cell's own genetic information, ina multicellular organism.

However, researchers have also observed cases of non-cell-autonomy,wherein one cell influences the phenotype of another cell. In amulticellular organism, different cells communicate with each other, andthus they influence and coordinate each other's behaviour and phenotype.In some cases, a mutation present in the genome of one cell may eveninfluence the phenotype of another cell that does not have said mutationin its genome.

A good example of non-cell-autonomy has been observed in plants. In thephloem sap of plants, many protein-coding mRNA transcripts, small RNAmolecules and proteins have been detected that can move from onelocation in the plant to another location. Thus, in some instances onecell produces an mRNA transcript, and then exports it to another cell,where the mRNA transcript may be translated into a protein, and wherethe protein fulfils its function. This exchange of information over longdistances, from one cell-type, tissue or organ to another via thevascular tissue, is very poorly understood, but it provides a new levelof complexity in how plants function, how they coordinate their owndevelopment and growth, and how their organs exhibit a concertedresponse to the plant's environment. It reveals that cells and organs indifferent parts of the organism communicate with each other, not only bymeans of small signalling molecules such as hormones, but also byexchanging macromolecules in a coordinated manner. The mode of action ofthis system (RNA motifs that trigger mobility, the extent of theirtransport, and their potential to be translated into functional proteinsafter transport) is however unknown in the prior art.

In the research leading to the present invention, it was observed thatmany mRNA transcripts that are present in the phloem sap of plants (andthat are enriched in the pool of mRNA transcripts that move acrosschimeric graft junctions) comprise a tRNA-like structure (TLS). It wasdemonstrated that tRNA-like structures are sufficient to mobilise mRNAtranscripts, by showing that mRNAs harbouring distinctive TLS move fromtransgenic roots into wild-type leaves and from transgenic leaves intowild-type flowers or roots, when wild-type and transgenic plants aregrafted onto each other. It was furthermore shown that these mobile mRNAtranscripts are translated into proteins after their transport. It wasalso found that bicistronic mRNA::tRNA transcripts (i.e. mRNAtranscripts harbouring a TLS) are frequently produced in Arabidopsisthaliana, and that they are enriched in the population of graft-mobilemRNAs. “Graft-mobile mRNAs” are mRNAs that are capable of moving acrossa graft junction.

RNA molecules are arguably the most functionally diverse biologicalmacromolecules found in cells, and their diverse roles are determined byboth their complex three-dimensional structure and by their primarysequence. An additional biological role of tRNA sequences (or tRNA-likestructures, TLSs) has now been uncovered in plants: they harbour a motifmediating mRNA transport to distant plant cells. Interestingly,transcript mobility was induced by tRNA^(Met) and tRNA^(Gly), but not byone particular tRNA^(Ile), which correlates with the absence of thistRNA^(Ile) in pumpkin phloem sap. As the present results indicate thattranscript mobility is mediated by a particular RNA structure, a tRNAmotif-scanning algorithm did indeed reveal that a significantly highnumber of identified mobile mRNAs harbours a TLS motif or is transcribedby genes in close proximity of annotated tRNA genes which seem tofrequently produce bicistronic poly (A)-RNA::tRNAs.

While the functional role of many mobile mRNAs in distant tissuesremains to be elucidated, evidence supports the notion that traffickingof small si/miRNA and large mRNAs via the phloem plays an important rolein regulating plant development. A surprisingly high number of mRNAs ispresent in phloem exudates and was shown to move across graft junctions,but no general and easily predictable RNA motif or conserved sequencemediating mobility could be identified in the graft-mobile transcriptpopulations. However, the present invention now demonstrates that asignificant fraction of mobile mRNAs carries a TLS motif that mediatesmobility across graft junctions.

Messenger RNA transfer does not strictly follow the source to sinkphloem flow, as it could be demonstrated that GUS::tRNA fusions not onlymove from shoot (source) to root (sink), but also vice versa. Twotransport pathways were found to exist for delivering mRNA molecules.One is based on passive, non-selective delivery from source to sink viathe phloem vessels. However, according to the invention also anotherpathway in form of a targeted and active transport system was found.This pathway inter alia mediates the delivery of mRNAs from root toshoot. Presence of an active mRNA delivery mechanism is supported by twofindings according to the invention, namely that sequences derived fromTLS are sufficient to confer mobility to heterologous mRNAs, and thatdeletion of a TLS in the plant endogenous CK1::tRNA^(Gly) bicistronictranscript, which is naturally mobile, makes it immobile.

It was shown according to the invention that TLSs or closely related RNAstructures mediate transport of a number of graft-mobile transcripts,and that specific tRNA sequences such as tRNA^(Gly)-, tRNA^(Met)-, andtRNA^(Met)-derived sequences trigger transport of otherwise non-mobiletranscripts, and that a significant number of mobile mRNAs harbour a TLSmotif.

The invention is thus based on the fact that this structural motif (TLS,with predicted stem-bulge-loop structures that are identical or verysimilar to a tRNA) plays a role in the coordinated long-distancetransport of mRNA transcripts.

In animals, evidence for long-distance intercellular mRNA transport alsoexists. In human plasma, for example, the presence of cell-free mRNA ofthe hTERT and MUC1 genes has been found to correlate with gastric cancer(Tani et al., 2007, Anticancer Res. 27: 1207-1212). Both genes have beenlinked to oncogenesis in humans. It is conceivable that the occurrenceof cell-free mRNA of these genes in plasma represents a mode ofcommunication between cancer cells and healthy cells. If healthy cellshave a means of uptake for these mRNA transcripts, they may startproducing the encoded proteins. Mammalian cells have been reported to becapable of taking up exogenous mRNA (see, for example, Probst et al.,2007, Gene Ther. 14: 1175-1180). Various mutations in hTERT have beenlinked to an increased risk of various cancers, and by sending out mRNAtranscripts comprising such mutation, one single cell carrying anoncogenic mutation in its genome (e.g. as the result of a non-inheritedde novo mutation) may have the capacity to distribute its mutatedversion of the mRNA transcript to many other cells that may not harboursaid mutation in their own genome, but that are receptive for taking upsaid transcript from the extracellular matrix. In such a manner, healthycells will produce a mutant (and potentially oncogenic) form of theencoded protein, even when their own, endogenous gene coding for thatprotein is not mutated. In the case of MUC1, it has been shown thatup-regulation of this gene is associated with cancer (Zaretsky et al.,2006, Mol. Cancer 5: 57). Here again, the presence of mRNA transcriptsof this gene in blood plasma would provide an opportunity for cells totake up these cell-free mRNA transcripts and to produce the proteinsencoded thereby, independently of the promoter activity of their own,endogenous MUC1 gene. This may lead to a higher than normal productionof this protein in many cells in the plasma, which, in turn, may belinked to oncogenesis.

The occurrence of disease-specific mRNA transcripts in blood has alsobeen observed in, for example, patients with the neurodegenerativedisorder Alzheimer's disease (Koh et al., 2014, Proc. Nat. Acad. Sci.USA 111: 7361-7366). This mRNA secretion from neurons may represent away in which this disorder progresses and spreads between the neurons ofa patient.

In animal cells, proteins and RNA may be secreted from cells throughvesicles in an unconventional secretion mechanism, which is differentfrom the Endoplasmic Reticulum (ER)/Golgi pathway. It is possible thatTLSs play a role in the selective loading of mRNA transcripts intovesicles and/or in the specific secretion of mRNA from cells into theextracellular matrix (e.g. blood, plasma). If the presence of a TLS alsoenables an mRNA transcript to be taken up by other cells, said TLS wouldessentially mediate cell-to-cell movement of mRNA transcripts inanimals, similarly to what has been observed in plants in the researchleading to the present invention.

The invention thus relates to a method for changing the intercellularmobility of an mRNA of a gene in an organism, comprising:

-   -   modifying a tRNA-like structure (TLS) present in the mRNA by        mutating the gene from which the mRNA is transcribed, or    -   including the sequence of a tRNA-like structure (TLS) in the        transcribed part of the gene.

There are essentially three ways in which the present invention can beapplied to change the intercellular mobility of an mRNA of a gene in anorganism. On the one hand, the mobility of an mRNA transcript that ismobile in a wild-type organism can be prevented, by modifying thetRNA-like structure that is naturally present in said mRNA transcript insuch a way that it no longer promotes mobility of the mRNA transcript.

On the other hand, an mRNA transcript that is not mobile in a wild-typeorganism can be made mobile, by providing said mRNA transcript with oneor more tRNA-like structures that have the ability to promote mobilityof the mRNA transcript. This embodiment can be applied to mRNAtranscripts from genes that are endogenous to the organism, but also tomRNA transcripts from genes that are not endogenous to the organism,such as genes that have been introduced into the organism's genome bymeans of genetic modification, or that have been introduced into theorganism in a transient manner, e.g. by injection, ingestion,transfection, or by exogenous application of mRNA molecules comprising aTLS on the organism's surface, and that are subsequently transportedfurther inside the organism and imported into cells by virtue of thepresence of said TLS.

In a third embodiment, the mobility of an mRNA transcript can be changedin the sense that the destination of the transcript inside the organism(i.e. the cell-type, tissue or organ where the mRNA transcript movestowards in a wild-type organism) changes as a result of modifications inthe TLS. In case different destinations in the organism are linked todifferent TLSs, modifying the original TLS of an mRNA can direct thetranscript to a different destination inside the organism, which resultsin an ectopic production of the encoded protein.

Modification of TLSs and inclusion of such TLSs in transcripts can beachieved by means of genetic modification. Genetic modificationcomprises transgenic modification or transgenesis, using a gene from anon-crossable species or a synthetic gene, and cisgenic modification orcisgenesis, using a natural gene, coding for a trait from the organismitself or from a sexually compatible donor organism.

In one embodiment, the organism of which the intercellular mobility ofthe mRNA of a gene is to be changed is a multicellular eukaryoticorganism, such as a plant, a fungus, or an animal. In a furtherembodiment, the organism is a dicotyledonous plant, a monocotyledonousplant, a gymnosperm, a bryophyte, an algae, a mammal, a non-mammalianvertebrate animal, or an invertebrate animal.

In the context of this invention, the term “organism” is intended to notonly comprise living multicellular plants, animals and fungi, but alsotissue cultures and cell cultures that have been derived from livingmulticellular plants, animals and fungi. Just like a living organism,said tissue cultures or cell cultures consist of multiple cells, betweenwhich cells mRNA can be exchanged.

According to a first aspect of the invention, said organism is a plant.In one embodiment, the organism is a plant belonging to one of thefollowing plant genera, or a cell culture or tissue culture derivedtherefrom: Allium, Apium, Beta, Brassica, Capsicum, Cichorium,Citrullus, Cucumis, Cucurbita, Benincasa, Daucus, Eruca, Lactuca,Lagenaria, Luffa, Phaseolus, Pisum, Lens, Raphanus, Solanum, Spinacia,Valerianella, Nicotiana, Petunia, Arabidopsis, Capsella, Arabis,Cardamine, Malus, Pyrus, Prunus, Vitis, Rosa, Fragaria, Populus, Fagus,Pinus, Picea, Ginkgo, Larix, Betula, Quercus, Salix, Alnus, Corylus,Amygdalus, Vaccinium, Rubus, Persea, Citrus, Castanea, Acer, Fraxinus,Coffea, Camellia, Theobroma, Olea, Cicer, Juglans, Pistacia, Arachis,Anacardium, Macadamia, Ficus, Litchi, Actinidia, Bougainvillea,Helianthus, Hibiscus, Malva, Glycine, Gossypium, Cannabis, Stevia,Opuntia, Ipomoea, Manihot, Humulus, Acacia, Medicago, Trifolium, Lotus,Vicia, Linum, Fagopyrum, Zea, Triticum, Avena, Hordeum, Oryza, Zizania,Secale, Triticosecale, Sorghum, Bambusa, Dendrocalamus, Saccharum,Cymbopogon, Pennisetum, Panicum, Festuca, Lolium, Phleum, Poa,Miscanthus, Asparagus, Agave, Yucca, Cocos, Elaeis, Phoenix, Amaryllis,Narcissus, Aloe, Canna, Iris, Colchicum, Crocus, Gladiolus, Juncus,Lilium, Tulipa, Musa, Dendrobium, Phalaenopsis, Vanilla, Typha,Zingiber, Curcuma, Lemna. Cell cultures and tissue cultures derived fromplants include, for example, microspore cultures, pollen cultures,callus cultures, root cultures, meristem cultures, protoplast cultures,mesophyll cell cultures, tissue or organ explants, and the BY-2 cellculture.

According to a second aspect of the invention, said organism is ananimal. In one embodiment, said organism is a mammal, a non-mammalianvertebrate animal, or an invertebrate animal. In a further embodiment,the organism is a mammal belonging to one of the following genera, or acell culture or a tissue culture derived therefrom: Mus, Homo, Rattus,Pan, Cricetus, Mesocricetus, Cavia, Canis, Oryctolagus. Mammalian cellcultures often used in research are, for example, CHO, HeLa, HEK293,HL-60, J558L, JY, K562, KBM-7, RBL, and COS-1.

In one embodiment, the intercellular mobility is between cells in thesame organ of an organism, or between cells in the same tissue cultureor cell culture. In this context, the concept of an “organ” alsocomprises, for example, the blood and lymph of animals. In anotherembodiment, the intercellular mobility is between cells in differentorgans of an organism.

Intercellular mobility of an mRNA of a gene in an organism may occuracross the plasma membranes of neighbouring cells in a tissue, or it mayoccur across the plasma membranes of cells that are not positionedadjacent to each other in a tissue. In the latter embodiment, saidmobility may take advantage of the organism's own means forlong-distance transport and cell-to-cell communication and/or of theextracellular matrix, such as the vascular tissue (phloem and/or xylem)or the apoplast in plants, or the blood stream or lymphatic system inanimals. Cells that are not positioned adjacent to each other in atissue may for example be cells that belong to different tissues ororgans in the organism, or cells that are not attached or connected toother cells, such as blood cells in an animal, pollen grains in aplant's anther, etcetera.

In the course of normal development and growth, the exchange of mRNAtranscripts between cells appears to be commonplace. It may however alsobe a phenomenon that plays an important role in the establishment and/orprogression of disease or in the organism's defence against disease, asit allows the transport of genetic material—in the form of an alreadytranscribed mRNA—from one cell to other cells, even across largedistances within an organism. This implies that the presence in anorganism of a single cell that harbours in a gene in its genome amutation that causes a certain effect in the protein encoded by saidgene, may result in the export of the mRNA comprising said mutation tomany other cells in the organism that—in their own genome—lack saidmutation. This may thus lead to a situation in which cells lacking amutation in their own genetic material import mRNA molecules comprisingsaid mutation, and subsequently translate that mRNA molecule into theprotein that is encoded by said mRNA molecule. The effect thereof isthat cells lacking said mutation may produce and harbour a protein thatcomprises said mutation. The mRNA transcripts of one mutated cell couldthus affect the behaviour and/or phenotype of a multitude of non-mutatedcells in the same organism, if the mRNA molecules sent out by saidmutated cell are mobile and are able to move from the cell by which theyhave been produced to another cell in the same organism where they canbe translated into protein. This mechanism may e.g. be relevant in theblood stream of animals, where essentially all cells are in a positionto communicate with each other by means of molecules that are present inthe blood, and in plants, wherein all organs are connected to each otherby means of the vascular tissue.

Alternatively, the one cell may not be mutated, but it may be subjectedto a stimulus, which causes it to produce a specific mRNA transcript ora specific splice-form of an mRNA transcript. This specific transcriptor splice-form thereof may subsequently be transported to other cells inthe organism, which had not (yet) received that stimulus, or which areincapable of responding to the stimulus. In plants, for example, cellsof a leaf may respond to a change in light quality, and signal thisperception to the roots. Root cells may detect changes in wateravailability in the soil, and communicate this to e.g. stomatal cells.Mobile mRNA transcripts may constitute a part of such intercellularcommunication signals between different parts of an organism.

In one embodiment, the invention relates to a method for changing theintercellular mobility of an mRNA of a gene in an organism, comprisingmodifying a tRNA-like structure present in the mRNA by mutating the genefrom which the mRNA is transcribed. Mutating the gene may comprisedeleting the sequence of the tRNA-like structure from the gene, mutatingthe sequence of the tRNA-like structure to change the tridimensionalconfiguration thereof, or inserting a genetic element (such as, forexample, a transposon, a retrotransposon, a T-DNA insertion, or aretroviral repeat sequence) into the gene to remove the tRNA-likestructure from the transcribed part of the gene, such that the sequenceencoding the TLS is no longer transcribed as part of the mRNA to whichit initially belonged, thus changing the mobility of that mRNA.

Deleting the sequence of the tRNA-like structure from the gene may, forexample, be accomplished by using genome editing tools such asCRISPR-Cas, using two guide RNAs designed to respectively targetsequences upstream and downstream of the coding sequence of thetRNA-like structure in the gene, thereby leading to double-strandedbreaks in the gene up- and downstream from the coding sequence of thetRNA-like structure in the gene, which may result in the removal of saidcoding sequence of the tRNA-like structure from the gene. Alternatively,deleting the sequence of the tRNA-like structure from the gene may beaccomplished in vitro, using standard molecular biology techniques orgene synthesis techniques, and subsequently introducing a DNA constructencoding a tRNA::mRNA bicistronic transcript, or the in vitrotranscribed transcript itself, into the organism.

The tridimensional configuration of a tRNA typically comprises stem-loopstructures: from 5′ to 3′ these are the D arm (or D stem-loop), the A oranticodon arm (or A stem-loop, or anticodon stem-loop), and the T arm(or TψC arm, TψC arm, or TψC stem-loop). Between the A and T arms avariable loop may be present. Mutating the sequence of the tRNA-likestructure to change the tridimensional configuration thereof maycomprise deleting part of the transcribed sequence thereof, mutating oneor more nucleotides involved in the formation of a stem-loop structurein the tRNA-like structure, or inserting one or more nucleotides in astem-loop of the tRNA-like structure.

Mutations may be induced at random or in a targeted manner, usingvarious mutagenesis methods known in the prior art. In vitro mutagenesismethods include, for example, site-directed mutagenesis, PCR-mediatedmutagenesis, and total gene synthesis. In vivo mutagenesis may, forexample, be achieved randomly by means of one or more chemicalcompounds, such as ethyl methanesulphonate, nitrosomethylurea,hydroxylamine, proflavine, N-methyl-N-nitrosoguanidine,N-ethyl-N-nitrosourea, N-methyl-N-nitro-nitrosoguanidine, diethylsulphate, ethylene imine, sodium azide, formaline, urethane, phenol,ethylene oxide, and/or by physical means, such as UV-irradiation,fast-neutron exposure, X-rays, gamma irradiation, and/or by insertion ofgenetic elements, such as transposons, T-DNA, retroviral elements.Mutations may also be introduced specifically by means of homologousrecombination, oligonucleotide-based mutation induction, or targetedgenome editing methods such as TALEN, zinc-finger technology, orCRISPR-Cas.

In one embodiment, deleting part of the transcribed sequence of thetRNA-like structure in a gene comprises deleting the A and/or Tstem-loops from the tRNA-like structure. As is shown in Example 2,removal of the A and T stem-loop structures results in the abolition ofmobility of an mRNA transcript that is mobile when the A and T stem-loopstructures are present. The removal of the D stem-loop, of the D and Tstem-loops or of the D and A stem-loops from a gene did not abolish themobility of the same mRNA transcript.

It should be noted that the term “tRNA-like structure” as used in thisapplication not only comprises intact tRNA structures that are presentin the mRNA transcript of a gene (and which can be screened for usingthe tRNA motif scan approach, explained in Example 2), but alsoincomplete tRNA structures that lack one or more stem-loop structuresbut that still retain the ability to enable the intercellular mobilityof the mRNA transcript of said gene. The term “tRNA-like structure” asused in the context of the present invention thus also encompasses tRNAstructures that lack the D stem-loop, the D and T stem-loops, or the Dand A stem-loops. The efficacy of such incomplete structures forconferring mobility to an mRNA transcript has been shown in Example 2and FIG. 4C.

In one embodiment, the present invention relates to a method forchanging the intercellular mobility of an mRNA of a gene in an organism,wherein the change in mobility results in a loss of the function of thegene. This is the case for genes whose mRNA transcript is transcribed inone cell-type, tissue or organ of an organism, but whose gene product(the encoded protein) functions in at least one other cell-type, tissueor organ of the organism, where its mRNA transcript is not produced. Forsuch genes, the intercellular mobility of their mRNA transcript is thusessential for the normal functionality of the gene in the organism. Theloss of intercellular mobility of the mRNA transcript of such a generesults in a loss of the function of the gene in the cells, tissues ororgans towards which the mRNA transcript moves during normal growth,development and/or functioning of the organism and in which it isnormally translated into protein. The loss of intercellular mobility ofthe mRNA transcript thus leads to the absence or reduced presence of thegene product (i.e. the encoded protein) in the cells, tissues or organswhere the mRNA transcript moves towards during normal growth,development and/or functioning of the organism. The phenotype of saidcells, tissues or organs may be affected by this absence or reducedpresence, and the morphology, responsiveness and/or functionality ofsaid cells, tissues or organs may be affected thereby. This situation isillustrated in Example 3, which shows that the loss of transcriptmobility for the CK1 gene in plants results in the absence of CK1transcript in the phloem, and in a very similar phenotype as could beobserved in a knock-out mutant that lacked the CK1 transcriptaltogether.

In another embodiment, the present invention relates to a method forchanging the intercellular mobility of an mRNA of a gene in an organism,comprising including the coding sequence of a tRNA-like structure in thetranscribed part of the gene.

In one embodiment, the present invention relates to a method forchanging the intercellular mobility of an mRNA of a gene in an organism,wherein the change in mobility results in ectopic presence of the geneproduct in the organism. In this manner, an mRNA transcript that doesnot display intercellular mobility can be rendered mobile, by includinga tRNA-like structure in said transcript.

In another embodiment, the present invention relates to a method forchanging the intercellular mobility of an mRNA of a gene in an organism,wherein the gene is a non-endogenous gene in at least part of theorganism. This situation is illustrated in Example 2, wherein theaddition of a TLS to the 3′UTR of the 1-glucuronidase-encoding gene(GUS) makes the GUS transcript mobile, and leads to translation of theprotein in distant parts of the plant, across a graft junction. Thissituation is also illustrated in Example 1, wherein a modified versionof DMC1 is provided in the form of a DNA construct in a transgenicrootstock. The modification comprise the addition of a TLS in the 3′UTRof the gene, and a truncation of the gene which turns the encodedprotein into a dominant-negative version that interferes with thefunction of wild-type DMC1. Said modified transcript is demonstrated tobe transported towards the male spores in a grafted scion, where theencoded protein (which is a dominant-negative version of DMC1)subsequently affects development.

Suitably, including the coding sequence of a tRNA-like structure in thetranscribed part of the gene is achieved by introducing thecomplementary sequence of the tRNA-like structure in a DNA constructcomprising the complementary sequence of the mRNA, or by introducing thecomplementary sequence of the tRNA-like structure in the endogenousgene, by means of e.g. genome editing tools such as CRISPR-Cas.

In one embodiment, the coding sequence of a tRNA-like structure isintroduced into the 3′ untranslated region (3′UTR) of the mRNA of thegene. Preferably, the coding sequence of a tRNA-like structure is thenintroduced after the stop-codon of a gene, such that it is present inthe transcribed part of the gene but does not affect the open readingframe of the gene. Alternatively, the coding sequence of a tRNA-likestructure may also be introduced into the 5′ untranslated region (5′UTR)of the mRNA of the gene, or in the open reading frame of the gene, insuch a way that the coding sequence is not disrupted or the readingframe is shifted.

The DNA construct may be stably integrated in the genome of theorganism, transiently expressed in the organism, or in vitrotranscribed. Said DNA construct comprises a suitable promoter that iscapable of driving expression of the gene in a suitable environment.There are, for example, promoters that are ubiquitous, organ-, tissue-or cell-type specific, and promoters that are constitutive or regulatedby environmental (physical or biological) or endogenous (developmental)queues, or regulated by chemicals.

Suitable constitutive promoter control sequences for mammals include,but are not limited to, cytomegalovirus immediate early promoter (CMV),simian virus (SV40) promoter, adenovirus major late promoter, Roussarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter,phosphoglycerate kinase (PGK) promoter, elongation factor (ED1)-alphapromoter, ubiquitin promoters, actin promoters, tubulin promoters,immunoglobulin promoters, fragments thereof, or combinations of any ofthe foregoing.

Suitable constitutive promoter control sequences for plants include, butare not limited to, 35S cauliflower mosaic virus (CaMV) promoter, opinepromoters, ubiquitin promoters, actin promoters, tubulin promoters,alcohol dehydrogenase promoters, fragments thereof, or combinations ofany of the foregoing.

Examples of suitable regulated promoter control sequences for animals orplants include, without limit, those regulated by heat shock, cold,drought, heavy metals, steroids (such as dexamethasone, β-estradiol),antibiotics, or alcohols (such as ethanol).

Non-limiting examples of tissue-specific mammalian promoters include B29promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter,desmin promoter, elastase-1 promoter, endoglin promoter, fibronectinpromoter, Flt-1 promoter, GFAP promoter, GPllb promoter, ICAM-2promoter, INF-13 promoter, Mb promoter, Nphsl promoter, OG-2 promoter,SP-B promoter, SYN1 promoter, and WASP promoter.

Non-limiting examples of tissue-specific plant promoters include theβ-phaseolin, glutenin, a-Kaf, zein, β-conglycinin and AGPase promoters(for seed-specific expression), the LAT52, GEX2, MAN5 and FRK4 promoters(for pollen-specific expression), the rolD, REO, Prx and Tip2 promoters(for root-specific expression), and the Zmglp1, PnGLP and PDX1 promoters(for leaf-specific expression).

The promoter sequence can be wild type or it can be modified for moreefficient or efficacious expression. In one exemplary embodiment, theencoding DNA can be operably linked to a CMV promoter for constitutiveexpression in mammalian cells.

In certain embodiments, the sequence of an endogenous or non-endogenousgene can be operably linked to a promoter sequence that is recognised bya phage RNA polymerase for in vitro mRNA synthesis. In such embodiments,the in vitro-transcribed RNA can be purified for administration to anorganism. For example, the promoter sequence can be a T7, T3, or SP6promoter sequence or a variation of a T7, T3, or SP6 promoter sequence.In an exemplary embodiment, the DNA encoding the fusion protein isoperably linked to a T7 promoter for in vitro mRNA synthesis using T7RNA polymerase.

In alternative embodiments, the sequence of an endogenous ornon-endogenous gene can be operably linked to a promoter sequence for invitro expression in bacterial or eukaryotic cells.

The DNA construct or the in vitro synthesised RNA transcript may beintroduced into the organism by various methods known in the prior art.

When a DNA construct is intended for stable integration in the genome ofan organism or for transient expression in an organism, it can beintroduced into said organism or into a cell derived therefrom by meansof (micro)injection, ingestion, transfection, electroporation,DEAE-dextran treatment, lipofection, nanoparticle-mediated transfection,protein transduction domain mediated transduction, virus-mediated genedelivery, PEG-mediated transfection of protoplasts, infection with asuitable virus or bacterium (such as Agrobacterium for plants),etcetera.

When a DNA construct is intended for in vitro transcription of theencoded mRNA, in vitro transcription may be achieved using any in vitrotranscription system known in the art. Alternatively, transcription mayalso be done in a heterologous organism, after which the transcript ofinterest is subsequently purified for further use.

An in vitro synthesised RNA transcript may be introduced into theorganism or into a cell derived therefrom by the methods listed above(such as injection into the blood stream of a mammal or into the phloemof a plant), and also by means of, for example, exogenous application onthe surface of the organism. Exogenous application may for example beperformed according to the disclosure in patent applicationWO2015/200539. Exogenous application to cell cultures or tissue culturesmay, for example, be performed using electroporation, transfection,etcetera.

When an in vitro synthesised RNA transcript is introduced into anorganism, a suitable promoter is not required, because transcriptiondoes not need to take place inside the organism's cells. Said transcriptmay immediately be translated, and/or it may move into cells of theorganism and from one cell of the organism to other cells of theorganism. According to the invention the movement of the transcript isdue to the presence of TLS.

Another application is the use of cultured cells that produce an mRNAtranscript harbouring a TLS for in vivo application in an organism. Forexample, cultured cells harbouring a gene that—when transcribed—givesrise to an mRNA transcript harbouring a TLS, may be injected (orotherwise administered) into a mammal's blood stream, where said cellssecrete an mRNA transcript harbouring a TLS into the extracellularmatrix. This mRNA transcript can subsequently be taken up by other cellsin the organism, due to the presence of a TLS. This constitutes anefficient way of delivering mRNA transcripts in vivo. Said mRNAtranscript may encode any protein that can be administered to theorganism. In a further embodiment, said mRNA transcript encodes atherapeutic protein, an endogenously occurring protein or a modifiedversion thereof, or a non-endogenously occurring protein. Suitably, theadministration of said cells to the organism is for the treatment of adisease, or for gene therapy.

In one embodiment, the complementary sequence of a tRNA-like structureis introduced into the transcribed part of a gene in a DNA construct,and the gene comprising the tRNA-like structure is transcribed in vitro.The mRNA transcript comprising the tRNA-like structure is subsequentlyintroduced into an organism, cell culture or tissue culture, such thatit can move into cells, and from one cell into other cells. Thisembodiment represents an application of the present invention that doesnot require the use of DNA constructs in vivo. The manner in which themRNA transcript comprising the tRNA-like structure is introduced into anorganism, cell culture or tissue culture, depends on the type oforganism, cell culture or tissue culture that is being used. Means bywhich introduction can be achieved comprise, for example, injection intothe blood stream of animals, injection into the vascular tissue orapoplast of plants, or exogenous application on the surface of theorganism. The use of cell-penetrating peptides may for examplefacilitate the entry of the mRNA transcript into the organism.

As stated above, the term “tRNA-like structure” as used in thisapplication not only comprises intact tRNA structures that are presentin the mRNA transcript of a gene (and which can be screened for usingthe tRNA motif scan approach, explained in Example 2), but alsoincomplete tRNA structures that lack one or more stem-loop structuresbut that still retain the ability to enable the intercellular mobilityof the mRNA transcript of said gene. The term “tRNA-like structure” asused in the context of the present invention thus also encompasses tRNAstructures that lack the D stem-loop, the D and T stem-loops, or the Dand A stem-loops. The term “tRNA-like structure” as used in thisapplication thus encompasses intact tRNA structures and tRNA structuresthat comprise at least the A stem-loop and/or the T stem-loop.

The efficacy of such incomplete structures for conferring mobility to anmRNA transcript has been shown in Example 2 and FIG. 4C. In oneembodiment, the tRNA-like structure is selected from the groupconsisting of tRNA^(Ala), tRNA^(Arg), tRNA^(Asn), tRNA^(Asp),tRNA^(Cys), tRNA^(Gln), tRNA^(Glu), tRNA^(Gly), tRNA^(His), tRNA^(Ile),tRNA^(Leu), tRNA^(Lys), tRNA^(Met), tRNA^(Phe), tRNA^(Pro), tRNA^(Ser),tRNA^(Thr), tRNA^(Trp), tRNA^(Tyr), tRNA^(Val). In another embodiment,the tRNA-like structure is selected from the group consisting oftRNAAla, tRNAArg, tRNAAsn, tRNAAsp, tRNACys, tRNAGln, tRNAGlu, tRNAGly,tRNAHis, tRNAlle, tRNALeu, tRNALys, tRNAMet, tRNAPhe, tRNAPro, tRNASer,tRNAThr, tRNATrp, tRNATyr, tRNAVal and it lacks the D stem-loop, the Dand T stem-loops, or the D and A stem-loops.

In a further embodiment, the tRNA-like structure comprises a tRNAanticodon with a sequence from 5′ to 3′ in the transcribed mRNA selectedfrom the group consisting of AGC, CGC, UGC, ACG, CCG, CCU, UCG, UCU,GUU, GUC, GCA, CUG, UUG, CUC, UUC, ACC, CCC, GCC, UCC, GUG, AAU, AAG,CAA, CAG, GAG, UAA, UAG, CUU, UUU, CAU, GAA, AGG, CGG, UGG, AGA, CGA,GCU, GGA, UGA, AGU, CGU, UGU, CCA, GUA, AAC, CAC, UAC. In anotherembodiment, the tRNA-like structure comprises a tRNA anticodon with asequence from 5′ to 3′ in the transcribed mRNA selected from the groupconsisting of AGC, CGC, UGC, ACG, CCG, CCU, UCG, UCU, GUU, GUC, GCA,CUG, UUG, CUC, UUC, ACC, CCC, GCC, UCC, GUG, AAU, AAG, CAA, CAG, GAG,UAA, UAG, CUU, UUU, CAU, GAA, AGG, CGG, UGG, AGA, CGA, GCU, GGA, UGA,AGU, CGU, UGU, CCA, GUA, AAC, CAC, UAC and it lacks the D stem-loop, orthe D and T stem-loops.

As shown in Example 2, some tRNA-like structures exist that have atridimensional structure that does not promote intercellular mobility ofan mRNA transcript, such as tRNA^(Ile) with anticodon UAU.

In one embodiment, the present invention relates to a method forchanging the intercellular mobility of an mRNA of a gene in a plant,wherein said plant consists of a rootstock of a first plant upon which ascion of a second plant has been grafted, and wherein the intercellularmobility of an mRNA of a gene in the first and/or the second plant hasbeen changed. This embodiment has been illustrated in Examples 1-3, withArabidopsis and Nicotiana (tobacco).

This embodiment can be carried out with any plant species for whichgrafting is possible. Commercially relevant plants that can be graftedare, for example, plants belonging to the genera Apium, Beta, Brassica,Capsicum, Cichorium, Citrullus, Cucumis, Cucurbita, Benincasa, Daucus,Eruca, Lactuca, Lagenaria, Luffa, Phaseolus, Pisum, Lens, Raphanus,Solanum, Spinacia, Valerianella, Nicotiana, Petunia, Arabidopsis,Capsella, Arabis, Cardamine, Malus, Pyrus, Prunus, Vitis, Rosa,Fragaria, Populus, Fagus, Pinus, Picea, Ginkgo, Larix, Betula, Quercus,Salix, Alnus, Corylus, Amygdalus, Vaccinium, Rubus, Persea, Citrus,Castanea, Acer, Fraxinus, Coffea, Camellia, Theobroma, Olea, Cicer,Juglans, Pistacia, Arachis, Anacardium, Macadamia, Ficus, Litchi,Actinidia, Bougainvillea, Helianthus, Hibiscus, Malva, Gossypium,Cannabis, Stevia, Opuntia, and Ipomoea. This is of course anon-exhaustive list.

In general, many dicotyledonous plants can be grafted, whereasmonocotyledonous plants cannot be grafted. However, a method hasrecently been developed to graft recalcitrant species such as soybean(Glycine max) and commercially important species of the Poaceae family,such as maize (Zea mays), rice (Oryza sativa), wheat (Triticumaestivum), etcetera (T. Harada, European patent application EP2918161).For those species this embodiment of the method of the present inventioncan thus also be applied.

This embodiment has various distinct applications. Firstly, theintercellular mobility of an mRNA of an endogenous gene may be changed.This may be done by changing the encoding gene in the rootstock or inthe scion of a grafted plant, and it may either lead to a loss ofmobility for an mRNA transcript that is naturally mobile in the plant,or to a gain of mobility for an mRNA transcript that is naturallynon-mobile in the plant. In case of a gain of mobility, the mRNAtranscript may move across the graft junction. In case of a loss ofmobility, the rootstock or scion may be depleted of the mRNA transcript,which may lead to phenotypic effects. Changing the encoding gene can bedone in all manners that have been described above, such as mutagenesis,genome editing and/or the use of transgenes.

In a further embodiment, the phenotype of the rootstock and/or scion ischanged. This may for example be achieved by the movement of a mobilemRNA transcript across the graft junction, where it complements for theabsence of that mRNA transcript in either the rootstock or the scion dueto a mutation. This thus represents a method for mutant complementation,by grafting a mutant plant onto a wild-type plant and rescuing themutant phenotype by means of a mobile mRNA transcript that compensatesfor the absence of the corresponding endogenous mRNA transcript.

In addition to changing the intercellular mobility of an mRNA of anendogenous gene, it is of course possible to induce additional mutationsin said mRNA transcript, such as point mutations, or to induce othermodifications in said mRNA transcripts, such as insertions or deletions.Such additional mutations or modifications may, for example, have aneffect on the primary sequence of the encoded protein, and on theencoded protein's structure and/or function.

An example of this embodiment is presented in Example 1, wherein amodified (dominant-negative) form of DMC1 is used to interfere with DMC1function in male spores.

Secondly, in another embodiment, a non-endogenous gene may be renderedmobile in an organism. The use of non-endogenous genes, such as reportergenes, is common practice in molecular biology, but the expressionpattern of such non-endogenous genes depends entirely on the use of asuitable promoter, that is able to drive a gene's expression in theorganism. Suitable promoters have been discussed above in this text.This situation is illustrated in Example 2, with β-glucuronidase (GUS).

Another example of a non-endogenous gene for which it is interesting torender its mRNA transcript mobile in an organism is Cas9. Cas9 is anRNA-guided endonuclease that has the capacity to create double-strandedbreaks in DNA in vitro and in vivo, also in eukaryotic cells. It is partof an RNA-mediated adaptive defence system known as Clustered RegularlyInterspaced Short Palindromic Repeats (CRISPR) in bacteria and archaea.Cas9 gets sequence-specificity when it associates with a guide RNAmolecule, which can target sequences present in an organism's DNA basedon their sequence. Cas9 requires the presence of a Protospacer AdjacentMotif (PAM) immediately following the DNA sequence that is targeted bythe guide RNA. The Cas9 enzyme has been first isolated fromStreptococcus pyogenes (SpCas9), but functional homologues from manyother bacterial species have been reported, such as Neisseriameningitides, Treponema denticola, Streptococcus thermophilus,Francisella novicida, Staphylococcus aureus, etcetera. For SpCas9, thePAM sequence is 5′-NGG-3′, whereas various Cas9 proteins from otherbacteria have been shown to recognise different PAM sequences. Innature, the guide RNA is a duplex between crRNA and tracrRNA, but asingle guide RNA (sgRNA) molecule comprising both crRNA and tracrRNA hasbeen shown to work equally well (Jinek et al, 2012, Science 337:816-821). The advantage of using an sgRNA is that it reduces thecomplexity of the CRISPR-Cas9 system down to two components, instead ofthree. For use in an experimental setup (in vitro or in vivo) this is animportant simplification.

An alternative for Cas9 is, for example, Cpf1, which does not need atracrRNA to function, which recognises a different PAM sequence, andwhich creates sticky end cuts in the DNA, whereas Cas9 creates bluntends.

On the one hand, genetic modification techniques can be applied toexpress an RNA-guided endonuclease and/or guide RNAs in eukaryoticcells. One or more DNA constructs encoding an RNA-guided endonucleaseand at least one guide RNA can be introduced into a cell or organism bymeans of stable transformation (wherein the DNA construct is integratedinto the genome) or by means of transient expression (wherein the DNAconstruct is not integrated into the genome, but it expresses anRNA-guided endonuclease and at least one guide RNA in a transientmanner). This approach requires the use of a transformation vector and asuitable promoter for expression in said cell or organism. Organismsinto which foreign DNA has been introduced are considered to beGenetically Modified Organisms (GMOs), and the same applies to cellsderived therefrom and to offspring of these organisms. In importantparts of the worldwide food market, transgenic food is not allowed forhuman consumption, and not appreciated by the public. There is thereforea need for an alternative, “DNA-free” delivery method of CRISPR-Cascomponents into intact plants, that does not involve the introduction ofDNA constructs into the cell or organism.

In the prior art, introducing the mRNA encoding Cas9 into a cell ororganism has been described, after in vitro transcription from a DNAconstruct encoding an RNA-guided endonuclease, together with at leastone guide RNA. This approach does not require the use of atransformation vector and a suitable promoter for expression in saidcell or organism.

Another known approach is the in vitro assembly of ribonucleoprotein(RNP) complexes, comprising an RNA-guided endonuclease protein (forexample Cas9) and at least one guide RNA, and subsequently introducingthe RNP complex into a cell or organism. The GMO status of this approachis not yet clear, but the introduction of RNP complexes into intactorganisms is technically challenging. In animals and animal cell andtissue cultures, RNP complexes have been introduced by means of, forexample, injection, electroporation, nanoparticles, vesicles, and withthe help of cell-penetrating peptides. However, the RNP complexes canonly perform their function in cells where they have been introducedinto, and this limits the efficiency of this approach. There is a needfor a self-propagating system wherein the CRISPR-Cas components movebetween cells.

In plants, the use of RNPs has been demonstrated in protoplasts, forexample with polyethylene glycol (PEG) transfection (Woo et al., 2015,Nat. Biotech. 33: 1162-1164). However, the applicability of thistechnique depends entirely on the availability of protocols for theregeneration of entire plants from protoplasts. Such protocols are notavailable for all plants, and there is therefore a need for analternative delivery method of CRISPR-Cas components in plant cells andin intact plants.

A specific application of the present invention involves themobilisation of an RNA-guided endonuclease-encoding mRNA in a plant. Ashown in Example 1 with DMC1-encoding mRNA, mRNA transcripts that arepresent in the phloem of a plant are able to reach the plant's malespores. When a similar approach is taken with an RNA-guidedendonuclease-encoding mRNA, it is therefore not only possible to modifythe genome of a plant's somatic cells, but also the genome of its malegermline, in the presence of at least one suitable guide RNA. The lattercategory of genome modifications is heritable, as they can betransmitted to the next generation.

Suitably, in one embodiment, the RNA-guided endonuclease gene can beencoded by a DNA construct that is integrated into the genome of oneplant, onto which another plant is subsequently grafted. In this manner,the cells of the rootstock harbour a transgenic construct, but the cellsof the scion do not. However, when the mRNA transcript encoding theRNA-guided endonuclease comprises a TLS, it is rendered mobile and it isable to cross the graft junction, and the RNA-guidedendonuclease-encoding transcript can be delivered into cells of thescion, where the RNA-guided endonuclease protein can be translated. Inthis situation, cells of the scion produce a protein that is not encodedin their own genome, and this protein is capable of modifying achromosomal sequence in said cells, when at least one suitable guide RNAis present in said cells.

Alternatively, the mRNA transcript encoding the RNA-guided endonucleaseand comprising a TLS may be introduced into a plant by direct injection,for example into the vascular tissue, or by exogenous application, ashas been described above. Said transcript will then behave as if it hadbeen produced inside the plant, and it will be delivered into cells ofthe plant, where the encoded protein will be translated and where itwill perform its function. In this embodiment, grafting is not arequirement.

After said modification of a chromosomal sequence has taken place, thecells can be used to produce plants that harbour said modification intheir genome, using any plant regeneration method known in the art (suchas in vitro tissue culture).

In a preferred embodiment, the RNA-guided endonuclease-encoding mRNAtranscript is translated into the encoded protein inside male spores ofa plant, where the encoded protein may modify a chromosomal sequence inthe presence of at least one suitable guide RNA. The at least one guideRNA may, for example, be introduced into the cells by means of injectioninto the anther, the phloem or other parts of the plant,Agrobacterium-mediated transformation, or exogenous application, or itmay be encoded by a DNA-construct that is present in the cells of theplant that produces said male spores. Said spores may be produced by thescion of a grafted plant. It has been shown that many small RNAmolecules (of comparable size as a typical guide RNA) are mobile in thephloem and are able to move systemically throughout the plant and acrossgraft junctions. It has also been shown that such small RNA moleculesare able to reach the male spores of a scion when they are transcribedin a rootstock (see for example patent application WO2013017683).

In a particular embodiment the invention thus provides a method formodifying a chromosomal sequence in a plant cell or spore, comprising:

a) combining a first plant comprising a rootstock, which harbours anucleic acid sequence encoding a modified, non-naturally occurringRNA-guided endonuclease protein, with a scion from a second plantgrafted onto the rootstock of the said first plant, whereby the saidnucleic acid sequence generates a transcript in the rootstock of thefirst plant, which transcript is transported systemically across thegraft junction to enter the scion of the second plant, and whichtranscript is imported into cells of the scion of the second plant,where it is translated into a functional protein;

b) providing at least one guide RNA or DNA encoding at least one guideRNA to at least one cell or spore of the scion of the second plant,wherein the at least one guide RNA is a single molecule comprising a 5′region that is complementary to a target site in a chromosomal sequenceof said second plant;

c) allowing each guide RNA to direct an RNA-guided endonuclease proteinto a targeted site in the chromosomal sequence, where the RNA-guidedendonuclease protein introduces a double-stranded break in the targetedsite, and allowing the cell to repair the double-stranded break by aDNA-repair process such that the chromosomal sequence is modified in atleast one cell or spore of the scion of the second plant.

The nucleic acid sequence encoding a modified, non-naturally occurringRNA-guided endonuclease protein may be a transgene that is stablyintegrated or transiently expressed.

In a particular embodiment, the RNA-guided endonuclease protein isderived from a Cas protein. The Cas protein is suitably Cas9, or avariant thereof. The Cas protein is for example derived from the genusStreptococcus.

In a particular embodiment, the nucleic acid sequence encoding amodified, non-naturally occurring RNA-guided endonuclease proteincomprises a tRNA-like structure that allows the transcript to betransported systemically across the graft junction from the rootstock ofthe first plant into the scion of the second plant. The nucleic acidsequence encoding a modified, non-naturally occurring RNA-guidedendonuclease protein may further comprise at least one localisationsignal to a DNA-containing organelle and optionally a marker domain.Non-limiting examples of marker domains are fluorescent markers (such asGreen Fluorescent Protein, Yellow Fluorescent Protein, mCherry), epitopetags (such as FLAG, His, HA, calmodulin), etcetera.

The localisation signal is suitably selected from the group comprising anuclear localisation signal, a chloroplast targeting signal, amitochondrial targeting signal.

The nucleic acid sequence encoding a modified, non-naturally occurringRNA-guided endonuclease protein may optionally further comprises a FokIendonuclease domain or another protein domain.

The tRNA-like structure can be tRNA^(Ala), tRNA^(Arg), tRNA^(Asn),tRNA^(Asp), tRNA^(Cys), tRNA^(Gln), tRNA^(Glu), tRNA^(Gly), tRNA^(His),tRNA^(Ile), tRNA^(Leu), tRNA^(Lys), tRNA^(Met), tRNA^(Phe), tRNA^(Pro),tRNA^(Ser), tRNA^(Thr), tRNA^(Trp), tRNA^(Tyr), tRNA^(Val). The term“tRNA-like structure” as used in the context of the present inventionalso encompasses tRNA structures that lack the D stem-loop, the D and Tstem-loops, or the D and A stem-loops.

The tRNA-like structure is located in the 3′UTR of the transcript, inthe 5′UTR of the transcript, or in the coding sequence of thetranscript.

In one embodiment, the guide RNA is a single-chain guide RNA, whereinthe guide RNA is a DNA-targeting RNA comprising a) a first segmentcomprising a nucleotide sequence that is complementary to a chromosomalsequence in a plant cell or spore, and b) a second segment thatinteracts with an RNA-guided endonuclease protein. Suitably, the guideRNA is a single-chain guide RNA comprising a crRNA and a tracrRNA.

Expression of a transgenic nucleic acid sequence in the first plant issuitably achieved by operably linking the said nucleic acid sequence toa promoter sequence that confers a ubiquitous expression profile or atissue- or cell-type specific expression profile onto the transgenicnucleic acid sequence, and/or to an inducible promoter. Suitablepromoters have been discussed above.

In one embodiment, the promoter sequence confers onto the transgenicnucleic acid sequence an expression profile that encompasses roots.

When expression of the transgenic nucleic acid sequence in the firstplant is transient, transient expression is suitably achieved by use ofan inducible promoter, selected from the group comprising heat-induciblepromoters, cold-inducible promoters, chemical-inducible promoters,steroid-inducible promoters and alcohol-inducible promoters. Suitablepromoters have been discussed above.

Providing the at least one guide RNA or DNA encoding the at least oneguide RNA to at least one cell or spore of the scion of the second plantis suitably accomplished by means of injection, Agrobacterium-mediatedtransformation, exogenous application, or stable integration ortransient expression of a DNA-construct.

The invention further relates to such a method, wherein exogenousapplication of the at least one guide RNA or DNA encoding the at leastone guide RNA comprises applying onto the scion of the second plant or apart thereof a mixture comprising:

a) a cationic polyelectrolyte;

b) an osmolyte; and

c) the at least one guide RNA or DNA encoding the at least one guideRNA, wherein the at least one guide RNA is a single molecule comprisinga 5′ region that is complementary to a target site in a chromosomalsequence of said second plant. Exogenous application may be performedaccording to the disclosure in patent application WO2015/200539.

In one embodiment of this latter method a chromosomal sequence of atleast one cell or spore of the second plant is genetically modifiedwithout the insertion of external genetic material.

In a particular embodiment of this latter method, the first plant andthe second plant belong to the same plant family. Suitably, the firstplant and the second plant belong to the same genus, in particular tothe same plant species.

In a further embodiment, the plant belongs to one of the followinggenera: Beta, Brassica, Capsicum, Cichorium, Citrullus, Cucumis,Cucurbita, Benincasa, Daucus, Eruca, Lactuca, Lagenaria, Luffa,Phaseolus, Pisum, Raphanus, Solanum, Spinacia, Valerianella, Nicotiana,Petunia, Arabidopsis, Capsella, Arabis, Malus, Pyrus, Prunus, Vitis,Rosa, Fragaria, Populus, Fagus, Pinus, Picea, Ginkgo, Larix, Betula,Quercus, Salix, Alnus, Corylus, Amygdalus, Vaccinium, Rubus, Persea,Citrus, Castanea, Acer, Fraxinus, Coffea, Camellia, Theobroma, Olea,Cicer, Juglans, Pistacia, Arachis, Anacardium, Macadamia, Ficus, Litchi,Actinidia, Bougainvillea, Helianthus, Hibiscus, Malva, Glycine,Gossypium, Cannabis, Stevia, Opuntia, or Ipomoea.

In an embodiment of this invention where grafting is not performed (forexample when the guide RNA and the RNA-guided endonuclease-encodingtranscript are both administered to the plant by means of a DNA-freedelivery method, such as, for example, injection of the guide RNA andthe RNA-guided endonuclease-encoding mRNA transcript into the phloem ofthe plant), the plant may belong to one of the following genera: Allium,Apium, Beta, Brassica, Capsicum, Cichorium, Citrullus, Cucumis,Cucurbita, Benincasa, Daucus, Eruca, Lactuca, Lagenaria, Luffa,Phaseolus, Pisum, Lens, Raphanus, Solanum, Spinacia, Valerianella,Nicotiana, Petunia, Arabidopsis, Capsella, Arabis, Cardamine, Malus,Pyrus, Prunus, Vitis, Rosa, Fragaria, Populus, Fagus, Pinus, Picea,Ginkgo, Larix, Betula, Quercus, Salix, Alnus, Corylus, Amygdalus,Vaccinium, Rubus, Persea, Citrus, Castanea, Acer, Fraxinus, Coffea,Camellia, Theobroma, Olea, Cicer, Juglans, Pistacia, Arachis,Anacardium, Macadamia, Ficus, Litchi, Actinidia, Bougainvillea,Helianthus, Hibiscus, Malva, Glycine, Gossypium, Cannabis, Stevia,Opuntia, Ipomoea, Manihot, Humulus, Acacia, Medicago, Trifolium, Lotus,Vicia, Linum, Fagopyrum, Zea, Triticum, Avena, Hordeum, Oryza, Zizania,Secale, Triticosecale, Sorghum, Bambusa, Dendrocalamus, Saccharum,Cymbopogon, Pennisetum, Panicum, Festuca, Lolium, Phleum, Poa,Miscanthus, Asparagus, Agave, Yucca, Cocos, Elaeis, Phoenix, Amaryllis,Narcissus, Aloe, Canna, Iris, Colchicum, Crocus, Gladiolus, Juncus,Lilium, Tulipa, Musa, Dendrobium, Phalaenopsis, Vanilla, Typha,Zingiber, Curcuma, Lemna.

The present invention will be further illustrated in the Examples thatfollow and that are not intended to limit the invention in any way. Inthe Examples reference is made to the following figures.

FIGS. 1A-1G. Dominant-negative DMC1 as a reporter construct causes malesterility in flowers.

(FIG. 1A) Schematic drawing of the _(DN)DMC1 RNA fusion constructs used.A. thaliana _(DN)DMC1 codes for a truncated protein lacking theN-terminal 92 amino acids and dominantly interferes with meiosis,resulting in misshaped pollen and partial male sterility. The _(DN)DMC1coding sequence was fused to graft-mobile StBEL5 sequences or phloemtRNA^(Met) at the 3′UTR to evaluate their potential to trigger _(DN)DMCmRNA transport over graft junctions.

(FIG. 1B) to (FIG. 1E) Fertile anthers of wild-type Nicotiana tabacumplants show regular pollen production with minimal abnormally shapedpollen (2-3%), whereas hpDMC1 siRNA transgenic tobacco plants producehigh numbers of abnormally shaped pollen and are sterile as previouslydescribed (Zhang et al., 2014). YFP-_(DN)DMC1 transgenic plants havenormal pollen production similar to wild type. Transgenic plantsexpressing _(DN)DMC1 fused with tRNA^(Met) or StBEL5 at the 3′UTRexhibit increased male sterility.

(FIG. 1C) and (FIG. 1E) Propidium iodide-stained pollen grains harvestedfrom transgenic plants were imaged by Confocal Laser Scanning Microscopy(CLSM) and evaluated by an automatic imaging analysis algorithm to countabnormally shaped pollen (Zhang et al., 2014), indicated by % numbers.Arrows indicate normal pollen; arrowheads indicate abnormally shapedpollen. Scale bars: 30 μm.

(FIG. 1F) and (FIG. 1G) Scheme of performed stem-grafts to evaluatetransport of mRNA to wild-type flowers.

FIGS. 2A-2G. _(DN)DMC1 fusion transcript transport induces aberrantpollen formation.

(FIG. 2A) Flowers of grafted wild-type stock plants supported by35S_(pro):YFP-_(DN)DMC1 transgenic scions and reciprocal grafts arefertile.

(FIG. 2B) Upper panel: Grafted wild-type/wild-type orwild-type/_(DN)DMC1::tRNA^(Met) plants showed normal pollen productionwhen mock-treated. Lower panel: Estradiol-induced expression of_(DN)DMC1::tRNA^(Met) in scion or stock plant parts resulted inpartially sterile anthers in both transgenic and wild-type flowers. Thelatter suggests _(DN)DMC1::tRNA^(Met) mRNA transport into, andexpression of the truncated DMC1 protein in, wild-type male meiocytes.

(FIG. 2C) Flowers of grafted _(DN)DMC1::StBEL5 transgenic plants. Upperpanel: Mock-treated wild-type/estradiol>>_(DN)DMC1::StBEL5 grafts showedweak male sterility. Lower panel: Flowers of grafted plants treated withestradiol exhibit partial male sterility.

(FIG. 2D) RT-PCR assays on RNAs samples from grafted wild-type tissuesrevealed that the YFP-_(DN)DMC1 control transcript is not allocated overgraft junctions into wild-type stock leaves (n=6) or scion flowers(n=8). ACTIN2 (ACT2) specific RT-PCR was used as a positive control.

(FIG. 2E) RT-PCR assays on RNA samples from grafted plants._(DN)DMC1::tRNA^(Met) and _(DN)DMC1::StBEL5 is detected in transgenicand in wild-type scion flowers. Appearance of a specific PCR product insamples from grafted wild-type stock leaves and wild-type flowers (redasterisks) suggests mobility of the _(DN)DMC1::tRNA^(Met) fusiontranscript. Number of tested grafted plants is shown on the right.

(FIG. 2F) CLSM images of sepals formed on YFP-_(DN)DMC1 producingscions. Upper panel: YFP-_(DN)DMC1/YFP-_(DN)DMC1 control graft withexpected high green fluorescence emitted by YFP-_(DN)DMC1. Middle panel:Control graft with siRNA-producing stock plants (hpDMC1) with expectedlow YFP fluorescence and distribution in YFP-_(DN)DMC1 flowers (Zhang etal., 2014). Lower panel: YFP-_(DN)DMC1 scion grafted onto_(DN)DMC1::tRNA^(Met) transgenic stock shows similar YFP fluorescencelevels as YFP-_(DN)DMC1/YFP-_(DN)DMC1 control grafts. Note thatYFP-_(DN)DMC1 fusion protein is detected in all epidermal leaf cellsexcept when grafted with hpDMC1 producing DMC1 siRNA lines. Greenindicates presence of YFP-_(DN)DMC1; Blue: plastid auto-fluorescence.Scale Bar: 300 μm.

(FIG. 2G) Statistical analysis of misshaped pollen appearing on graftedplants. Misshaped pollen formation was significantly higher on wild-typescions supported by _(DN)DMC1::tRNA^(Met) and _(DN)DMC::StBEL5 stockplants than in control grafts. Asterisks indicate highly significantdifferences against controls using Chi-square test for independence ofvariables in a contingency table. Biological replicates: n>8. Error barsindicate S. D. For details see Table 1.

FIGS. 3A-3D. tRNA^(Met)::_(DN)DMC1 movement into flowers and pollenphenotype.

(FIG. 3A) Upper panel: Flowers of non-grafted transgenic plantssupported by _(DN)DMC1::tRNA^(Met) and tRNA^(Met)::_(DN)DMC1 are fertilewhen mock-treated. Lower panel: Application of estradiol inducing_(DN)DMC1::tRNA^(Met) and tRNA^(Met)::_(DN)DMC1 expression resulted inpartially sterile anthers, suggesting production of thedominant-negative _(DN)DMC1 protein.

(FIG. 3B) Upper panel: Grafted tRNA^(Met)::_(DN)DMC1/wild-type orwild-type/tRNA^(Met)::_(DN)DMC1 plants showed partial sterile pollenproduction when mock-treated. Lower panel: After application ofestradiol onto grafted tRNA^(Met)::_(DN)DMC1/wild-type orwild-type/tRNA^(Met)::_(DN)DMC1 plants formation of aberrantpollen/sterile anthers in wild-type flowers suggesttRNA^(Met)::_(DN)DMC1 mRNA transport and expression of the truncatedDMC1 protein in wild-type male organs.

(FIG. 3C) RT-PCR assays on poly(A)-RNA samples from transgenic_(DN)DMC1::tRNA^(Met) (n=6) and tRNA^(Met)::_(DN)DMC1 (n=7) tissuesindicate presence of fusion transcripts in both transgenic and inwild-type scion flowers (red asterisks) suggesting mobility of thetRNA^(Met)::_(DN)DMC1 fusion transcript over graft junctions. The numberof grafted plants tested is shown on the right. ACTIN2 (ACT2) specificRT-PCR was used as a positive control.

(FIG. 3D) Statistical analysis of misshaped pollen appearing on graftedplants (Table 1). Compared to mock-treated control grafts, production ofmisshaped pollen was significantly higher in estradiol-treated_(DN)DMC1::tRNA^(Met) and tRNA^(Met)::_(DN)DMC1 transgenic plants and inwild-type scions supported by tRNA^(Met)::_(DN)DMC1. Asterisks indicatestatistically significant differences against controls using Chi-squaretest of independence of variables in a contingency table. Biologicalreplicates: n>8. Error bars indicate S.D. For details see Table 1. Notethat >3 independent transgenic _(DN)DMC1::tRNA^(Met) ortRNA^(Met)::_(DN)DMC1 lines were used in the grafting experiments.

FIGS. 4A-4D. GUS::tRNA fusion transcripts and mobility in graftedArabidopsis thaliana.

(FIG. 4A) Schematic drawing of used 35S_(pro):GUS::tRNA fusionconstructs (for sequences of tRNA^(Met) tRNA^(Gly), tRNA^(Ile), andtRNA^(Met) deletions see FIG. 5).

(FIG. 4B) Example of a hypocotyl grafted GUS::tRNA^(Met)/wild-type(Col-0) plant. Blue colour indicates presence of GUS activity in thehypocotyl above the graft junction (arrow) and in the wild-type roottip.

(FIG. 4C) GUS activity in leaves and primary root tips detected inGUS::tRNA/wild-type grafts. The numbers indicate the fraction of GUSstaining detected in the wild-type root tips or wild-type leafvasculature (arrows) of plants grafted with the indicated transgenicline. At least 3 independent transgenic lines were used for each graftcombination (for additional images of grafted plants see FIGS. 6A-6B).

(FIG. 4D) RT-PCR on poly(A)-RNA samples harvested from grafted plants.Three samples from 3-5 grafted plants were pooled and tested forpresence of GUS transcripts in wild-type tissue (asterisks). Numbersindicate occurrence of GUS poly(A) RNA in the tested wild-type root orwild-type leaves RNA samples. ACTIN2 (ACT2) specific RT-PCR was used asa positive control confirming mRNA presence in the samples.

FIG. 5. tRNA sequences fused to the 3′UTR of GUS.

Alignment of tRNA sequences fused to the GUS coding DNA sequence. TheGUS sequence harbouring an intron was fused with tRNA^(Met) (CAU),tRNA^(Gly) (GCC), tRNA^(Ile) (TAT) and deletion variants of tRNA^(Met)(CAU) named tRNA^(Met) ΔD, ΔDA, ΔDT, and ΔAT. The length of tRNAsequences (counted from the GUS ATG start codon) is indicated at theright. Asterisks indicate the GUS TGA stop codon. Red boxes indicate theanticodon sequence of the cloned tRNAs.

FIGS. 6A-6B. Images of hypocotyl-grafted wild-type/GUS::tRNA transgenicplants.

(FIG. 6A) Example images of Arabidopsis thaliana Col-0 plants that werehypocotyl-grafted. The presented wild-type (Col-0)/transgenic plantgraft combinations are indicated above. Magnified images of leaf areas,root tips, and graft junction (arrows) are indicated by orangerectangles. Blue coloured tissues in the shown plant parts indicatepresence of GUS activity.

(FIG. 6B) Micrographs of thin sections made on a paraffin embeddedgrafted plant. Wild type was grafted with GUS::tRNA^(Met) ΔDT transgenicstocks. GUS enzymatic activity (blue colour) was detected in vascularcells of wild-type leaves and wild-type hypocotyl in cells associated tothe vasculature (arrows). Arrowheads indicate xylem vessels.

FIGS. 7A-7G. Mobile A. thaliana mRNAs and occurrence of tRNA-likemotifs.

(FIG. 7A) Number of all identified mobile transcripts (n=3630) withpredicted tRNA-like structures found by the default RNAMotif tRNAdescriptor, which does not capture the tRNA^(Ile) (TAT) (FIGS. 8A-8C).Absolute counts as well as enrichment in relation to transcripts notfound in the mobile database are shown. Asterisks indicate significantcounts (p<0.05) according to Fisher's exact test.

(FIG. 7B) Normalised frequency (estimated density) and cumulativerelative frequency (ecdf) of inter-gene distances of tRNA-mRNA tandemgene pairs with the tRNA being located within 1000 nucleotides up- ordownstream of genes coding for mobile transcripts (blue) or non-mobilepredicted transcripts (grey). Vertical dashed lines indicate medians ofshown distributions. Mobile transcript encoding loci in comparison toloci not producing mobile transcript show a significantly closerproximity to tRNA genes (two-sample one-sided Kolmogorov-Smirnov test,p<0.004; Cohen's D d=0.349).

(FIG. 7C) Number of tRNA genes according to their anticodon, which weredetected as poly(A)-RNA::tRNA bicistronic transcripts in RNA-Seq data.The distribution of the 94 tRNA genes observed bicistronically are givenin orange; distribution of the 24 tRNA genes associated with mobiletranscripts are shown in blue. The total of TAIR10 annotated tRNA genesis presented in gray. tRNA genes with bicistronic transcripts confirmedby RT-PCR are indicated by asterisks, experimentally tested tRNA fusionsare marked by arrows. (FIG. 7D) Schematic diagram of A. thaliana CHOLINEKINASE 1 (CK1, AT1G71697) gene and analysed insertion mutants. Note thatCK1 mRNA exists as a bicistronic poly(A)—tRNA transcript and that theck1.2 mutant harbours a T-DNA insertion between the CK1 stop codon andthe annotated tRNA^(Gly) (AT1G71700) in the 3′ UTR resulting in atruncated poly(A) transcript lacking the tRNA^(Gly) sequences. RT-PCRprimers are indicated as follows: P1 binding to exon 7 of CK1 CDS and P3binding to tRNA^(Gly) sequences were used for wild-type CK1::tRNA^(Gly)identification. P1 together with P2 which is stretching the T-DNA leftborder and CK1 3′UTR were used for specific ck1.2 detection.

(FIG. 7E) RT-PCR with the indicated primers revealed that the CK1poly(A) transcript is present in ck1.2 mutant samples (asterisks)originating from stem-grafted wild-type Col-0 tissue. In the reciprocalwild-type samples a mutant CK1 poly(A) transcript produced in ck1.2 andlacking the 3′UTR tRNA^(Gly) sequence was not detected.

(FIG. 7F) Phenotype of ck1.1 and ck1.2 and quantitative Real Time RT-PCRof transcripts. Wild-type and ck1.2 plants show similarly high levels ofCK1 expression whereas ck1.1 mutants show very low expression. Rosettearea size measurements on adult plants revealed that both ck1.1 andck1.2 mutants are significantly smaller than wild type (Students T-test,mutant vs. wild type; p-value ck1.1=0.006; p-value ck1.2=0.002; n=16plants/line). Error bars: S.D.

(FIG. 7G) Schematic folding structure of the GUS TLS 3′UTR motifspredicted according to their minimal free energy (MFE). Structures areshown for GUS::tRNA^(Met) (SEQ ID NO: 111), GUS::tRNA^(Met) ΔD/Tloop/stem (“ΔD/T loop/stem”; SEQ ID NO: 112), GUS::tRNA^(Met) ΔA/Tloop/stem (“ΔA/T loop/stem”; SEQ ID NO: 113), GUS::tRNA^(Met) ΔDloop/stem (“ΔD loop/stem”; SEQ ID NO: 114), GUS::tRNA^(Met) ΔD/Aloop/stem (“ΔD/A loop/stem”; SEQ ID NO: 115), GUS::tRNA^(Gly(GCC)) (SEQID NO: 116), and GUS::tRNA^(Ile(TAT)) (SEQ ID NO: 117).

FIGS. 8A-8C. Computational analysis of tRNA-like sequences (TLSs)present in mobile mRNAs.

(FIG. 8A) tRNA structure descriptor and structural alignment oftRNA^(Met), tRNA^(Gly), and tRNA^(Ile). The numbers indicate theaccepted descriptor ranges of structural elements (stem and loops). Notethat the tRNA descriptor does not recognise the structure of thetRNA^(Ile) which did not confer mobility when fused to GUS.

(FIG. 8B) Evaluation of parameter space for the tRNA descriptorconstraints. Given a tRNA descriptor model, we tested for enrichment ofTLS in mobile relative to non-mobile predicted gene transcripts. Thedefault tRNA descriptor (indicated by a red circle at coordinate origin,p=0.001) indicates an enriched number of hits in mobile transcripts incomparison to non-mobile transcripts. Departure from the default tRNAdescriptor parameters is expressed by the sum over differences of theconstraint values for the minimum element length (y-axis) as well as bythe sum over the maximum element length differences (x-axis). Out of20,000 produced descriptors, 13,598 that have less than 5,000 gene hitsto the non-mobile dataset (excluding tRNA genes) were plotted. Colouringis according to p-values resulting from the counts of motif hits tomobile versus non-mobile transcript sequences (Fisher's exact test),point sizes indicate the number of hits to mobile transcripts.Increasing minimum stem and loop lengths in the RNA structure modelsresult in lower numbers of matches in the mobile transcript dataset.Similarly, relaxing maximum length constraints results in larger numberof false positive matches in the non-mobile dataset and, thus, motifhits to mobile versus non-mobile transcripts get less significant.

(FIG. 8C) Degree of structuredness associated with the predictedstructure of the 150 nt 3′-terminal sequence region of mobile (n=3,606)vs. non-mobile (n=23,132) predicted mRNAs. Upper graph showsstructuredness in terms of number of predicted paired bases. Lower graphin terms of predicted minimum free energy (MFE). The vertical dashedlines denote the medians of the two distributions.

EXAMPLES Example 1

tRNA^(Met) Fusion Transcripts Move into Flowers

Introduction

To establish a simple phenotypic scoring system for mRNAs harbouringpredicted mobility motifs such as TLSs, a dominant-negative A. thalianaDMC1 variant (_(DN)DMC1) was used that lacks the N-terminal 92 aminoacid residues (FIG. 1A) and that interferes with meiosis progression.

Methods

Plant Material and Growth Conditions

Tobacco (N. tabacum cv. Petite Havana) plants were grown under asepticconditions on agar-solidified medium containing 30 g L⁻¹ sucrose. Rootedtobacco plants were transferred to soil and grown to maturity understandard greenhouse conditions (relative humidity: 55%; day temperature:25° C.; night temperature: 20° C.; diurnal cycle: 16 h light/8 hdarkness; light intensity: 190-600 μE·m⁻²·s⁻¹).

Grafting and Estradiol Treatment

Tobacco plants used for grafting experiments were grown two to threemonths on soil in the greenhouse. A standard splice grafting procedurewas used as previously described (Zhang et al., 2014). In short, plantswith the same stem diameter carrying five fully expanded leaves wereused as stock and scion material; rootstocks were prepared by removingthe apical leaves from the top of the plant and keeping two to threesource leaves. Scions were prepared by cutting the stem 3 to 4 cm belowthe apex and removing the source leaves. A long slanting cut was made onthe rootstock stem (about 30 degrees from vertical) with a matching cutat the scion base. The surfaces of both cuts were immediately pressedtogether and the junction was tightly wrapped with parafilm. The firstweek after grafting the scion was covered with a plastic bag and keptunder high humidity.

After the graft junction was established, axillary branches and leavesemerging at the stock were removed in order to enforce apical dominanceof the scion. Before flower induction, 5 μM 17-β-estradiol mixed withLanolin (Sigma-Aldrich) (1000× stock solution: 5 mM 17-β-estradiol inDMSO, stored at −20° C.) was applied with soaked tissue paper onto theadaxial side of stock plant leaf surfaces to induce gene expression. Thetissue paper was left on the surface to mark the site of induction.After flowers appeared on the scion, part of the induced leaf andemerging first closed flowers were sampled for fusion transcriptpresence by RT-PCR.

RNA Isolation and Reverse Transcription Reactions

Samples were prepared in 1 mL Trizol reagent (Invitrogen) (0.5 ml/100 mgtissue) as described previously (Zhang et al., 2009). Aftercentrifugation (10,000 g, 10 minutes at 4° C.), the supernatant (˜1 ml)was transferred to a new RNase-free tube and extracted once with 200 μland once with 50 μl chloroform. To precipitate the RNA the supernatantwas supplemented with two volumes of 99% isopropanol, 0.1 volumes of 3 Msodium acetate (pH 5.2), 1 μg of linear acrylamide (Invitrogen), andincubated >1 h at −20° C. After centrifugation (16,000 g, 30 minutes at4° C.), the resulting pellet was washed twice with 80% ethanol, oncewith 99% ethanol, air dried, and resuspended in 20 μl RNase-free water.To determine RNA quality and concentration, 1 μl of each RNA sample wassubmitted to agarose gel electrophoresis (2%, agarose, 1×TBE) andquantified using a NanoDrop ND-1000 (Thermo Scientific).

Reverse transcription reaction was performed with 1 U/μl AMV reversetranscriptase (Promega) with following modifications: total RNA (˜4 μg)was denatured at 70° C. for 10 minutes in the presence of oligo (dT)primer followed by a 5 minutes annealing incubation at 37° C. prior tothe RT-reaction, then incubated at 42° C. for one hour, and 72° C. for10 minutes for deactivation. RT-PCR was conducted under standard PCRconditions with 40-45 cycles. Oligonucleotides used for RT-PCR arelisted in Table 2.

TABLE 2 Oligonucleotides used in the study. SEQ Purpose/Size ofConstruct Primer sequences ID NO PCR fragment _(DN)DMC1::StBEL5FK868-F 5′-GATGCTCCGAATCTCGCTGA-3′   1  491 bpFK869-R 5′-GTTGCTTGCTGCTGGTGAAG-3′   2 _(DN)DMC1::_(Met)tRNAFK868-F 5′-GATGCTCCGAATCTCGCTGA-3′   3  260 bpFK851-R 5′-TTCCGCTGCGCCACTCTGATT-3′   4 DMC1::_(Met)tRNAFK868-F 5′-GATGCTCCGAATCTCGCTGA-3′   5  260 bpFK851-R 5′-TTCCGCTGCGCCACTCTGATT-3′   6 _(DN)DMC1::ZmKN1HDFK868-F 5′-GATGCTCCGAATCTCGCTGA-3′   7  495 bpFK779-R 5′-TAGAAGGCATTGGTGGTG-3′   8 _(Met)tRNA::_(DN)DMC1FK774-F 5′-ATAACCCACAGGTCCCAG-3′   9  422 bpFK771-R 5′-TTCCATTCCCTCCTTTCA-3′  10 YFP-_(DN)DMC1FK938-F 5′-CCCGACAACCACTACCTGAG-3′  11  353 bpFK858-R 5′-TCATCGAGAGCTTGACACCCTGT-3′  12 NbActin (GB: X69885)FK422-F 5′-CACCGGTATTGTGTTGGACTC-3′  13  303 bpFK423-R 5′-AGGACCTCAGGACAACGGAAACG-3′  14 ACTIN2 (At3g18780)FK424-F 5′-GGAAGGATCTGTACGGTAAC-3′  15  245 bpFK425-R 5′-TGTGAACGATTCCTGGACCT-3′  16 BP primer for CHOLINEFK907-F 5′-ATTTTGCCGATTTCGGAAC-3′  17  504-804 bp KINASE 1 (CK1) T-DNAinsertion verification LP primer salk_023420FK908-R 5′-TGGTTCATTACAGGAGAACCG-3′  18 1096 bp RP primer salk_023420FK909-F 5′-TTTGTGAATCTCAGGGAATGC-3′  19 BP + RP 504-804 bpLP primer salk_070759 FK914-R 5′-AGCAGCCATCTCACAAAAGTG-3′  20 1012 bpRP primer salk_070759 FK915-R 5′-TCTAAAACGCGTTTTGCAAAC-3′  21 BP +RP 492-792 bp CK1-_(Gly)tRNA fusion FK883-F 5′-CTATGGGGAATCATCTCGGG-3′ 22  331 bp detection FK884-R 5′-CACTAGACCACTGGTGCTTC-3′  23CK1-_(Gly)tRNA detection FK883-F 5′-CTATGGGGAATCATCTCGGG-3′  24  392 bpFK992-R 5′-CCGTGGCAGGGTACTATCAT-3“  25 CK1-_(Gly)tRNA mobilityFK883-F 5′-CTATGGGGAATCATCTCGGG-3′  26  309 bpdetection binding to ck1.2 FK962-R 5′-GTAGACTATATATTGTGGTGTAAAC-  27(salk_023420) 3′ Construction GUS-tRNAFK963-F 5′-CTAG CCATGG TAGATCTGAGG-3′  28 fusion forward primerConstruction GUS- FK944-R 5′-GCG CGG TGA CCT GCA CCA GCC  29Size of GUS-Gly _(Gly)tRNA reverse primerGGG AAT TGA ACC CGG GTC TGT ACC GTG GCA tRNA 2141 bpGGG TAC TAT CAT GCC ACT AGA CCA CTG GTGCAA TTC ACA CGT GAT GGT GAT GGT G-3′ Construction GUS-FK945-R 5′-GCG CGG TGA CCT ATC AGA GCC ACC  30 Size of GUS-Met_(Met)tRNA reverse primer TT CGA TCC TGG GAC CTG TGG GTT ATG GGCtRNA 2141 bp CCA CCA CGC TTC CGC TGC GCC ACT CTG ATAATT CAC ACG TGA TGG TGA TGG TG-3′ Construction GUS-FK946-R 5′-GCG CGG TGA CCG CTT CCG GCG  31 Size of GUS-Met_(Ile)tRNA reverse primer GGG CTC GAA CCC GCG ACC TTC GGC TCA TAAtRNA 2142 bp GAC CAA CGC TCT AAC CAA CTG AGC TAC GGGAGC AAT TCA CAC GTG ATG GTG ATG GTG-3′ Construction GUS-FK948-R 5′-GCG CGG TGA CCT ATC AGA GCC  32 Size of GUS-Met_(Met)tRNA D loop deletion AGG TTT CGA TCC TGG GAC CTG TGG GTT ATGtRNA dD 2128 bp (dD) reverse primerGGC CCA CCA CCA CTC TGA TAA TTC ACA CGT GAT GGT GAT GGT G-3′Construction of GUS- FK950-R 5′-GCG CGG TGA CCT ATC AGA GCC  33Size of GUS-Met _(Met)tRNA D AnticodonAGG TTT C GAT CCT GGG ACC TGC CAC CAC TCT tRNA dDT 2113loop deletion (dDA) GAT AAT TCA CAC GTG ATG GTG ATG GTG-3′ bpreverse primer Construction GUS-FK949-R 5′-G CGC GGT GAC CTA TCA GAG CGG  34 Size of GUS-Met_(Met)tRNA D and TΨC loop ACC TGT GGG TTA TGG GCC CAC CAC CAC TCTtRNA dDA 2113 deletion (dDT) reverseGAC AAT TCA CAC GTG ATG GTG ATG GTG-3′ bp primer Construction GUS-FK947-R 5′-GC GCG GTG ACC TAT CAG AGC GGA  35 Size of GUS-Met_(Met)tRNA Anticodon and CCT GCC ACG CTT CCG CTG CGC CAC TCT GATtRNA dAT 2111 TΨC loop deletion (dAT) AAT TCA CAC GTG ATG GTG ATG GTG-3′bp reverse primer Adding MettRNA + XbaIFK951-R 5′-gcgc GGTGACC TCTAGA TATCAGAGC-  36 + BstEII reverse primer 3′Adding GlytRNA + XbaI FK952-R 5′-gcgc GGTGACC TCTAGA TGCACC-3′  37 +BstEII reverse primer Adding IleRNA XbaIFK953-R 5′-gcgc GGTGACC TCTAGA GCTTCCGG-3′  38 + BstEII reverse primerGUS mobility test FK1091-F 5′ CAACAGCTTCCGGACCGCAC-3′  39  428 bpFK1092-R 5′ GATTGAGCGCGATGACGTCA-3′  40 GUS-_(Met)tRNA mobilityFK1079-F 5′ CGAGTACTACCAGGCGAACC-3′  41  316 bp FK1081-R 5′CAGAGCCAGGTTTCGATCCTG-3′  42 GUS-_(Gly)tRNA mobility FK1079-F 5′CGAGTACTACCAGGCGAACC-3′  43  281 bp FK1086-R 5′ GCAGGGTACTATCATGCCAC-3′ 44 GUS-_(Ile)RNA mobility FK1080-F 5′ ACCACGTCGTGTTCGATGAG-3′  45 272 bp FK1085-R 5′ GGCTCATAAGACCAACGCTC-3′  46 Construction of GUS-FK1079-F 5′ CGAGTACTACCAGGCGAACC-3′  47  316 bp_(Met)tRNA D loop deletion FK1081-R 5′ CAGAGCCAGGTTTCGATCCTG-3′  48(dD) reverse primer GUS-_(Met)tRNA D and FK1080-F 5′ACCACGTCGTGTTCGATGAG-3′  49  255 bp TΨC loop deletion (dDT) FK1083-R 5′GTTATGGGCCCACCACCACTC-3′  50 mobility verification GUS-_(Met)tRNA D andFK1079-F 5′ CGAGTACTACCAGGCGAACC-3′  51  274 bp Anticodon loop deletionFK1084-R 5′ GATCCTGGGACCTGCCAC-3′  52 (dDA) mobility verificationGUS-_(Met)tRNA Anticodon FK1079-F 5′ CGAGTACTACCAGGCGAACC-3′  53  292 bpand TΨC loop deletion FK1082-R 5′ GCTGCGCCACTCTGATAATTC-3′  54(dAT) reverse primer GUS mobility FK1099-F 5′ AGAACGCTAGCCATCACCATC-3′ 55  127 bp Poly A primer FK1100-R 5′ GCCAAATGTTTGAACGATCGGG-3′  56GUS mobility FK1099-F 5′ AGAACGCTAGCCATCACCATC-3′  57  135 bppolyA primer FK1090-R 5′ GCAAGACCGGCAACAGGAT-3′  58GUS detection by QRT- FK1093-F 5′ CGCGTCCAAGGAAACAAGAAG-3′  59  142 bpPCR FK1094-F 5′ TTCACACGTGATGGTGATGGTGA-3′  60 Actin2 primer QRT-PCRFK1097-F 5′ TCCCTCAGCACATTCCAGCAGAT-3′  61   69 bp (AT3G18780)FK1098-F 5′ AACGATTCCTGGACCTGCCTCATC-3′  62 UBQ10 primer QRT-PCRFK1095-F 5′ CACACTTCACTTGGTCTTGCGT-3′  63   61 bp (AT4G05320)FK1096-R 5′ TAGTCTTTCCGGTGAGAGTCTTCA-3′  64 CK1 and mutant detectionFK1105-F 5′ ATCTTCTGGGGACTATGGGGA-3′  65  126 bp by QRT-PCR FK1106-R 5′TCATCCTTCAAGAAGCAAAGGC-3′  66 CK1 and mutant detection FK1107-F 5′TCATACACGCCAGAACTCTTTC-3′  67  198 bp by QRT-PCR FK1108-R 5′CCAACCGATACTTATCCATCTCTA-3′  68 AT3G01700-tRNA^(Pro)FK1122-F 5′-CCGGATTCTTCATCTTCTCTCTCT-3′  69  305 bpbicistronic poly A RT- FK1123-R 5′-  70 PCRCCTAAGCGAGAATCATACCACTAGACC-3′ tRNA^(Pro)-AT3G01710FK1124-F 5′-GTTACAGTAGCAGAGAGGTCTTACA-  71  280 bpbicistronic poly A RT- 3′ PCR FK1125-R 5′-CGAGTTCAATTCTCGGAATGCC-3′  72tRNA^(Pro)-AT3G01710 FK1126-F 5′-CAACAGACCAAACTAAGAAAGCTC-  73  305 bpbicistronic poly A RT- 3′ PCR FK1125-R 5′-CGAGTTCAATTCTCGGAATGCC-3′  74AT4G34030-tRNA^(Arg) FK1127-F 5′-  75  257 bp bicistronic poly A RT-CCTTTAGAAGATACTCGATTTGGTGTC-3′ PCRFK1128-R 5′-GTCTGATTAGAAGTCAGACGCCT-3′  76 tRNA^(Arg)-AT4G34040FK1129-F 5′-CGACATAAAAGCACCGTTCC-3′  77  514 bp bicistronic poly(A) RT-FK1130-R 5′-GGCCCAATGGATAAGGCGT-3′  78 PCR AT5G03740-tRNA^(Leu)FK1131-F 5′-CAGTGCAGCTGCTTGAGAAGA-3′  79  453 bp bicistronic poly A RT-FK1132-R 5′-GTCTTCCCCCTTAACCACTCG-3′  80 PCR tRNA^(Leu)-AT5G03740FK1133-F 5′-GGTTTGCCCGAGTGGTTAAG-3′  81  548 bp bicistronic poly A RT-FK1134-R 5′-GACAAGGTGCAGCTTCTTTGA-3′  82 PCR AT5G41600-tRNA^(Leu)FK1135-F 5′-CTCTGAACAAGAAGAAGGATTAAGG-  83  741 bpbicistronic poly A RT- 3′ PCR FK1136-R 5′-CCTTAGACCACTCGGCCATC-3′  84tRNA^(Leu)-AT5G41610, FK1137-F 5′-GACTTCTACGGATAAAGACTCTGA-3′  85 399 bp bicistronic poly A RT- FK1138-R 5′-TCTAAGGCGCCAGACTCAAG-3′  86PCR AT4G14410-tRNA^(Thr) FK1139-F 5′-GCCTCCTGCTGCTTAAACTCT-3′  87 277 bp bicistronic poly A RT- FK1140-R 5′-GTAAGCGGGAGGTCTTGAGT-3′  88PCR tRNA^(Thr)-AT4G14420 FK1141-F 5′-GCTCCAAAGGCAAAAGCAAAC-3′  89 430 bp bicistronic poly A RT- FK1142-R 5′-AACGGGTGCTCTAACCAACT-3′  90PCR AT2G33130-tRNA^(Met) FK1143-F 5′-GCTAGCGCGTAGGTCTCATA-3′  91  638 bpbicistronic poly A RT- FK1144-R 5′-GAAGCAAAGCTGCCGAGATG-3′  92 PCRAT2G33130-tRNA^(Met) FK1143-F 5′-GCTAGCGCGTAGGTCTCATA-3′  93  697 bpbicistronic poly A RT- FK1145-R 5′-TTCTCCACCGTCCATGCAAT-3′  94 PCRtRNA^(Met)-AT2G33150 FK1146-F 5′-CGCTCGCTAGAGAGGACCAT-3′  95  432 bpbicistronic poly A RT- FK1147-R 5′-GACCTACGCGCTAGCCAACT-3′  96 PCRAT4G27870-tRNA^(Gln) FK1148-F 5′-CTGAAACTGAATCTTGCCTGGAG-3′  97  284 bpbicistronic poly A RT- FK1149-R 5′-GGACTCTGAATCCAGTAACCCG-3′  98 PCRActin2 (At3g18780) FK1152-F 5′-ACTTTCATCAGCCGTTTTGA-3′  99  190 bpFK1153-R 5′-ACGATTGGTTGAATATCATCAG-3′ 100Expression Constructs

To produce a dominant-negative AtDMC1 with a N-terminal 92 amino aciddeletion in A. thaliana DMC1 (_(DN)DMC1) transcripts with 3′UTR and5′UTR fusions an expression binary constructs named pRD1 and pRD4 werecreated based on a pMDC7 (Curtis and Grossniklaus, 2003) backbone. The_(DN)DMC1 fragment was introduced 5′ or 3′ of the pMDC7 gateway cloningcassette which resulted in a template binary vector used to clone via agateway reaction the RNA sequences of StBEL5, or tRNA^(Met) between the_(DN)DMC1 ORF and promoter or terminator (FIG. 1A).

Synthetic oligonucleotides were used to produce gateway ENTRY cloneswith the according sequence for gateway recombination with the binaryvector (Table 2). The binary vector constructs based on pMDC7 allowestradiol-induced _(DN)DMC1::RNA or RNA::_(DN)DMC1 expression.pEarlyGate104 used for 35S_(pro):YFP-_(DN)DMC1 expression and the DMC1siRNA N. tabacum line (35S_(pro):BcDMC1 hpRNAi) and its function waspreviously described (Zhang et al., 2014).

Microscopy and Pollen Shape Analysis

The statistical pollen shape analysis indicating sterility was performedas described previously (Zhang et al., 2014). Tobacco pollen wascollected and stained with propidium iodide (0.01 mg/ml, MolecularProbes, USA). To image the shape and size of the pollen a ConfocalLaser-Scanning Microscope (CLSM; TCS SP5; Leica Microsystems) was used.The system had the following settings: Detection Channel 2 (red):570-650 nm. The Channel 2 gain (PMT) was set between 500-600 V, Pinhole:1.0 Airy Units, 5 Z-stacks with 5-6 μm were merged and used for theshape recognition algorithm as described (Zhang et al., 2014).

YFP fluorescence was detected as described (Zhang et al., 2014) withfollowing settings: Sequential channel scan mode with a maximum aerialpinhole of 1.5 Airy Units. To compare the YFP fluorescence intensitybetween plants the same settings such as laser power, gain voltage,pinhole, objective, magnification, and channel/filter wavelengths wereused. Z-stack images were assembled and processed using the Image Jsoftware package (NIH). Detection Channel 1 (green): 535 to 617 nm;Detection Channel 2 (red): not used; Detection Channel 3 (blue;Chloroplast/plastid auto-fluorescence): 695 to 765 nm. Channel 1 gain(PMT) was set between 500-600 V.

Results

DMC1 is a specific meiotic cell-cycle factor and a member of the highlyconserved RecA-type recombinase family of DNA-dependent ATPases activeduring meiosis in sporogenic cells. Lack of a functional DMC1/RAD51complex induces achiasmatic meiosis resulting in the formation ofanomalously shaped pollen containing an aberrant number of chromosomesand, consequently, is necessary for proper pollen development. Thus,production of misshaped pollen in anthers and decreased fertility serveas a readout, indicating the presence of mobile DMC1 siRNA (FIGS. 1B-1C)and/or the presence of a translation product of a mobiledominant-negative _(DN)DMC1 mRNA.

To implement an mRNA mobility reporter system, transgenic Nicotianatabacum plants were produced expressing _(DN)DMC1 mRNA fused to theknown mobile full-length StBEL5 transcript (Cho et al., 2015)(_(DN)DMC1::StBEL5) and a full-length tRNA^(Met) (AT5G57885;_(DN)DMC1::tRNA^(Met) tRNA^(Met)::_(DN)DMC1) (FIG. 1A) which wasdetected in the phloem sap of pumpkin. Plants expressing theseconstructs were verified to show a pollen sterility phenotype (FIGS.1D-1E) and they were used in grafting experiments (FIGS. 1F-1G) toevaluate transcript mobility from transgenic source tissue to wild-typeflowers.

It was first confirmed by RT-PCR that _(DN)DMC1 itself is not mobileand, thus, is suitable as a transcript mobility reporter producing apollen phenotype (FIG. 2A). Next, the mobility of the fusion transcriptswas addressed by grafting _(DN)DMC1::StBEL5, _(DN)DMC1::tRNA^(Met) ortRNA^(Met)::_(DN)DMC1 transgenic plants with wild-type plants, andpollen sterility and the presence of the fusion transcript in wild-typeflowers examined (FIGS. 2B-2G; FIGS. 3A-3D).

As expected, after induction with estradiol, the _(DN)DMC1::StBEL5scions grafted onto wild-type stocks showed a significantly higherpercentage of aberrant pollen formation in their flowers (30.9±7.6%)than grafted wild-type plants (4.0±3.0%) (FIG. 2C; Table 1). Confirmingthat StBEL5 fusion transcripts are mobile, wild-type plants grafted onto_(DN)DMC1::StBEL5 stocks produced a significantly higher number ofmisshaped pollen (19.7±14.3%) than wild-type controls and the presenceof fusion transcript was confirmed by RT-PCR in closed wild-type flowers(FIG. 2E). Thus, _(DN)DMC1—RNA fusion constructs can be employed as anRNA mobility reporter system by producing a quantifiable pollenphenotype.

Next, to learn whether a phloem-allocated tRNA contains the necessarystructural information mediating mRNA movement over long-distances,transgenic plants expressing the 3′UTR _(DN)DMC1::tRNA^(Met) (FIG. 2B)or the 5′UTR tRNA^(Met)::_(DN)DMC1 (FIGS. 3A-3D) fusion construct weregrafted. Expression was induced by applying estradiol to the transgenicsource leaves (stock) or transgenic stem (scion)—1.5 weeks aftergrafting and prior to flower induction.

Estradiol-treated grafted plants formed a significantly higher number ofmisshaped pollen (14.2+7.6%; Table 1) compared to control grafts andwild-type plants (FIGS. 2A-2B), and RT-PCR assays confirmed the presenceof _(DN)DMC1::tRNA^(Met) and tRNA^(Met)::_(DN)DMC1 poly(A) transcriptsin wild-type flowers formed on transgenic stock plants (FIG. 2E, FIGS.3A-3D).

TABLE 1 Pollen shape analysis on wild-type, transgenic, and graftedNicotiana tabacum plants. # of plants with # of visual pollen # pollensterility (# of independent analyzed % misshaped Scion/Stock plants)lines (# plants) pollen P-value Significance Controls hpDMC1 siRNA 10(10) 3 5101 (10) 78.08 ± 10.86% 0.0 *** wild type  0 (10) 3 5365 (10)7.51 ± 2.59% — ns YFP-_(DN)DMC1 0 (6) 3 947 (4) 3.27 ± 1.84% — nsControl Wild type/wild type  0 (12) 3 1187 (7)  4.04 ± 3.00% — ns grafts(before estradiol induction) Wild type/wild type 0 (6) 3 643 (6) 3.27 ±3.96% — ns (after estradiol induction) Wild type/YFP- 0 (8) 3 1068 (4) 5.62 ± 3.10% — ns _(DN)DMC1 YFP-_(DN)DMC1/ 0 (6) 2 3664 (5)  5.84 ±1.31% — ns wild type YFP-_(DN)DMC1/ 0 (6) 4 824 (3) 4.13 ± 2.83% — nsYFP-_(DN)DMC1 _(DN)DMC1::tRNA^(Met) Wild type/ 0 (6) 3 239 (3) 3.35 ±1.32% — ns grafts _(DN)DMC1::tRNA^(Met) (before estradiol induction)Wild type/ 11 (17) 8 1525 (6)  14.23 ± 7.60%  3.76e−24 ***_(DN)DMC1::tRNA^(Met) (before estradiol induction)_(DN)DMC1::tRNA^(Met/) 3 (6) 3 416 (3) 13.94 ± 2.28%  3.78e−10 *** wildtype (before estradiol induction) _(DN)DMC1::tRNA^(Met/) 16 (19) 9 2331(8)  38.65 ± 22.46%  7.52e−270 *** wild type (after estradiol induction)YFP-_(DN)DMC1/ 2 (5) 2 374 (1) 10.96 ± 0.00%  0.00417 **_(DN)DMC1::tRNA^(Met) (after estradiol induction) _(DN)DMC1::StBEL5 Wildtype/ 0 (4) 2 433 (2) 8.08 ± 0.76% 0.51261 ns grafts _(DN)DMC1::StBEL5(before estradiol induction) Wild type/ 10 (14) 4 1398 (10) 19.67 ±14.33% 3.60e−65 *** _(DN)DMC1::StBEL5 (after estradiol induction)_(DN)DMC1::StBEL5/ 3 (4) 2 243 (2) 21.40 ± 6.92%  9.04e−65 *** Wildtype/ (before estradiol induction) _(DN)DMC1::StBEL5/ 12 (14) 6 1200(5)  30.92 ± 7.56%   8.43e−221 *** Wild type/ (before estradiolinduction) tRNA^(Met)::_(DN)DMC1 Wild type/ 0 (4) 2 398 (2) 5.28 ± 2.02%— ns grafts tRNA^(Met)::_(DN)DMC1 (before estradiol induction) Wildtype/ 14 (21) 4 2291 (8)  14.80 ± 14.73% 2.42401e−27   ***tRNA^(Met)::_(DN)DMC1 (after estradiol induction) tRNA^(Met)::_(DN)DMC1/2 (4) 2 332 (3) 9.04 ± 1.78% 0.01835 * Wild type (before estradiolinduction) tRNA^(Met)::_(DN)DMC1/ 18 (22) 6 4540 (12) 26.81 ± 18.68% 1.34e−177 *** Wild type (after estradiol induction) Non-_(DN)DMC1:tRNA^(Met) 3 (6) 6 723 (2) 17.48 ± 5.83%  4.10e−22 *** grafted(before estradiol transgenic induction) plants _(DN)DMC1::tRNA^(Met) 6(6) 6 555 (4) 38.74 ± 15.32%  3.92e−115 *** (after estradiol induction)_(DN)DMC1::StBEL5 2 (5) 5 709 (4) 5.92 ± 2.61% — ns (before estradiolinduction) _(DN)DMC1::StBEL5 5 (5) 5 313 (3) 56.87 ± 6.24%   1.77e−175*** (after estradiol induction) tRNA^(Met)::_(DN)DMC1 3 (6) 6 637 (2)25.27 ± 9.91%  5.15e−56 *** (before estradiol induction)tRNA^(Met)::_(DN)DMC1 5 (6) 6 287 (3) 37.28 ± 4.45%  1.29e−84 *** (afterestradiol induction) Asteriks indicate statistical significanceregarding the enhanced number of misshaped pollen against wild-typecontrol using Chi-square test of independence of variables in acontingency table (ns—not significant, * p-value ≤ 0.05, ** p-value ≤0.01, *** p-value ≤ 0.001)

To exclude the possibility that the grafted chimeric plants produce amobile DMC1 siRNA silencing signal that moves into the wild-type flowertissues and triggers a pollen sterility phenotype, the_(DN)DMC1:tRNA^(Met) plants with a reporter line producing a yellowfluorescent YFP-_(DN)DMC1 fusion protein were grafted. In contrast tothe DMC1 siRNA control lines, no systemic siRNA mediated silencing ofthe YFP-_(DN)DMC1 reporter construct could be detected in sepals (FIG.2F). Thus, the _(DN)DMC1::tRNA^(Met) fusion transcript does not inducesystemic silencing, and the observed defects in pollen formation ingrafted plants (FIG. 2G) can be attributed to the systemic delivery ofthe _(DN)DMC1 fusion transcripts.

In summary, presence of the full-length tRNA^(Met) sequence in the 5′ or3′ UTR triggers transport of the _(DN)DMC1 poly(A) transcript from stockto source leaves and into sporogenic tissues, where it is translated asit interferes with meiosis in male tissues.

Example 2

tRNAs Harbour a Signal for Systemic mRNA Movement

To evaluate whether particular tRNA sequences related to viral TLSmediate systemic mRNA movement, the core tRNA sequences of thephloem-imported tRNA^(Met) (72 bases; TAIR #AT5G57885) and tRNA^(Gly)(74 bases; TAIR #AT1G71700), and the non-phloem imported tRNA^(Ile) (73bases; TAIR #AT3G05835) (Zhang et al., 2009) were fused to the 3′UTR ofthe cell-autonomous β-GUS mRNA sequence (FIG. 4A, FIG. 5). To evaluatethe mobility of the fusion transcripts, A. thaliana Col-0 linesexpressing 35S_(pro):GUS or 35S_(pro):GUS::tRNA fusion constructs wereproduced and hypocotyl-grafted with Col-0 wild type (shoot or root).

Methods

Plant Material and Growth Conditions

A. thaliana seeds of wild type (Col-0), and transgenic 35S_(pro):β-GUS,ck1.1 (SALK_070759), ck1.2 (SALK_023420) plants of ecotype Col-0 wereused and grown in controlled environmental chambers for growth assays oron soil in the greenhouse (relative humidity: 60%; day temperature: 22°C.; night temperature: 19° C.; diurnal cycle: 16 h light/8 h darkness;light intensity: 170-200 μE·m⁻²·s⁻¹). The SALK-lines were obtained fromthe Salk Institute Genomic Analysis Laboratory, California (Alonso etal., 2003).

Grafting

A. thaliana hypocotyl grafting was performed as described (Thieme etal., 2015). In short, plants were grown vertically on solid 0.5 MSmedium (1% sucrose) at 22° C. with a photoperiod of 8 h light (fluencerate of 100 μmol m⁻² s⁻¹). The temperature was increased to 26° C. 4days after germination to reduce adventitious root formation. 6 to 7days after germination seedlings were used for grafting under sterileconditions as described (Thieme et al., 2015).

In short, seedlings were cut transversely in the middle of the hypocotylwith a razor blade (Dumont; No. 5), and a silicon collar (NeoTecha; Ø0.30×0.60 mm) was slid over the stock in which the scion was inserted.Grafted plantlets were placed on solid 0.5 MS medium (supplemented with1% agar and 1% sucrose) and grown at 22° C. (8 h light). Appearingadventitious roots were cut every two days and after two weekssuccessfully grafted plants were submitted to histochemical GUS stainassays, or root and shoot materials were harvested separately for RT-PCRdetection of GUS transcripts.

Expression Constructs GUS fusion constructs harbouring tRNA^(Met) (AUG),tRNA^(Gly) (GGC), or tRNA^(Ile) (AUA) and tRNA^(Met) (AUG) variants inthe 3′UTR were created by PCR amplification using an NcoI GUS forwardprimer covering the GUS start codon and by a BstEII GUS reverse primercovering the GUS stop codon and the tRNA sequence. The resulting PCRfragment was amplified again with an unspecific XbaI reverse primerharbouring an XbaI site for identification of the cloned fragment. Theresulting NcoI-BstEII digested fragments were cloned into theaccordingly digested pCambia1305.1 (Chen et al., 1998) allowingexpression of the GUS::tRNA constructs driven by a 35S promoter. Allsynthetic oligonucleotides used in the PCR reactions are listed in Table2.

β-Glucuronidase (GUS) Detection

Histochemical reactions with substrate X-Gluc were performed with plantmaterial incubated in 80% Acetone for 20 minutes at −20° C., washed 2×with 50 mM NaPO₄ buffer pH 7.0. The staining solution (1 mM X-Glucdiluted in 25 mg/ml, in 50 mM NaPO₄ pH 7.0 buffer, supplemented with 2mM Potassium Ferricyanide, 2 mM Potassium Ferrocyanide, 0.1% TritonX-100) was vacuum infiltrated for 15 minutes. The staining reaction wascarried out at 37° C. overnight and stopped by rinsing the tissues threetimes in 70% ethanol for 1 h.

The stained plant material was examined by stereo-microscopy (Leica,DFC300, FX). For thin sections, GUS stained samples were dehydrated inan ethanol series including a fixation step 20% ethanol, 35% ethanol,50% ethanol, FAA prepared fresh (50% ethanol, 3.7% Formaldehyde, 5%acetic acid), 70% ethanol for 30 minutes each at room temperature. Thenthe samples were embedded in paraffin using the enclosed tissueprocessor Leica ASK300S and the embedding centre Leica EG1160. 10 μm and20 μm longitudinal and traverse sections were placed on polyL-Lysine-coated slides.

After drying the samples overnight at 42° C. the slides were de-waxedtwice in histoclear for 10 minutes and then incubated twice in 99.8%ethanol for 10 minutes under constant movement at room temperature.After drying overnight the cover slips were mounted with Entellan new(Merck Millipore) and examined by an epi-fluorescence microscope (BX61,Olympus).

Bioinformatic Analysis

tRNA Motif Scans

Reference sequences of all protein-encoding genes [available cDNAsequence data associated with all protein-coding Arabidopsis genes,TAIR10 (Lamesch et al., 2012), excluding organellar genomes] werepartitioned into distinct sets based on their annotation as mobile ornon-mobile as detected in heterografted Arabidopsis accessions orCuscuta-parasite Arabidopsis-host interactions (Thieme et al., 2015).Subsets were generated for genes common to both mobile sets (n=486),present in at least one of them (n=3606), as well as according to theobserved movement direction (root-to-shoot, shoot-to-root, andbidirectional). All genes and associated transcripts assigned asnon-mobile were used as controls. All sets were filtered for duplicatesequences, and annotated tRNA genes were removed. tRNA sequence datawere obtained from the tRNAdb (Juhling et al., 2009). Prior to structuremotif scans, each sequence was padded with 50 “N” leading and trailingcharacters to facilitate the detection of terminally located tRNAstructures without asymmetric ends at the tRNA acceptor arm which arerequired by the default tRNA descriptor. All sets were analysed byRNAMotif version 3.1.1 (Macke et al., 2001) using the provided tRNAstructure descriptor and default parameter settings. Motif enrichmentassociated with genes encoding mobile transcripts compared to backgrounddata was assessed by Fisher's exact test. Specificity of the searchedtRNA-like structure was assessed by permutation scans of the defaulttRNA descriptor.

20,000 different tRNA descriptors were produced by randomly altering theaccepted minimum and maximum lengths limits for the stems and thesingle-stranded loops in the model (normal distribution using μ=0,sigma=5; minimum stem length set to 3 nt). Each descriptor was evaluatedagainst the mobile/non-mobile data by RNAMotif with default settings.Structuredness; i.e. the percentage of base-paired nucleotides andassociated energetics, within the 3′UTR was addressed by excising the150 nt 3′-terminal sequence portion and subsequent analysis of itspredicted secondary structure (RNAfold, default settings).

tRNA-mRNA Tandem Scans

Genes adjacent to tRNA loci were identified according to TAIR10 genemodels including protein-coding, non-coding genes, and pseudogenes.Statistical significance of the difference of gene proximitydistributions (distances between tRNA-genes and mobile vs. non-mobilegene neighbours) was estimated by the non-parametric Kolmogorov-Smirnovtest (1-sided test); relevance was assessed by the effect size (Cohen'sD) based on the mean observed differences and associated standarddeviations.

Bicistronic tRNA Analysis

A. thaliana reference genome (TAIR10) sequence information was obtainedfrom The Arabidopsis Information Resource (www.arabidopsis.org),associated gene model descriptions (gtf version 10.30) were taken fromplants.ensembl.org. Paired-end RNA-Seq data (100nt reads from both ends)was retrieved from the Sequence Read Archive (SRA)(www.ncbi.nlm.nih.gov/sra), accessions SRX853394 (14.1G bases, rootsample) and SRX853395 (15.3G bases, shoot sample) (Thieme et al., 2015)as well as DRX014481 (19G bases, root sample) and DRX014482 (32.7Gbases, root sample). Read data were quality trimmed and Illumina adaptersequences were clipped by using Trimmomatic (Lohse et al., 2012)standard settings (ILLUMINACLIP:<adapterfile>:2:40:15, LEADING:3,TRAILING:3, SLIDINGWINDOW:4:15, and MINLEN:36).

Mapping of sequences mate pairs to the A. thaliana reference genome(TAIR10) was done by STAR v2.5.1 (Dobin et al., STAR: ultrafastuniversal RNA-seq aligner, Bioinformatics 2012) based on Ensembl genemodel descriptions. Considering the high number of tRNA genes in theArabidopsis genome and their similar sequences, reads with multiplealignments were excluded, minimum overhang for gene junctions was set to10 nt for annotated junctions and 20 nt for unannotated junctions,maximum number of allowed mismatches per pair was 10 nt(outFilterMultimapNmax 1, alignSJDBoverhangMin 10, alignSJoverhangMin20, outFilterMismatchNmax 10).

Subsequently, all read pairs mapping to chromosomes 1 to 5 with aminimum alignment quality Q>10 were checked to be intersecting withboth, tRNA and mRNA gene annotations. Finally, identified 132bicistronic poly(A)-RNA::tRNA transcripts were grouped by their tRNAgene identity (118 unique tRNA genes, FIG. 7C) as well as by theprotein-coding gene (120 unique genes) and the assigned transcriptmobility. Results were compared to the list of annotated tRNA-mRNAtandems and statistical significance for the observed overlap to thebicistronic transcripts was assessed by Fisher's exact test.

Results

Two weeks after grafting, GUS enzyme activity was visualised in situ(FIGS. 4B-4C; FIG. 6). Control grafts with transgenic 35S_(pro):GUSplants lacking the tRNA sequences in the 3′ UTR showed no GUS activityand GUS mRNA presence in distant wild-type root (n=0/55 grafts) or leaf(n=0/43 grafts) tissues, indicating that neither the GUS mRNA nor theGUS protein moves over grafting junctions (FIGS. 4C-4D).

However, GUS activity was detected in phloem-associated cells inwild-type roots after hypocotyl-grafted with transgenic scion plantsexpressing GUS::tRNA^(Met) (n=9/44 grafts) or GUS::tRNA^(Gly) (n=6/25grafts). No GUS activity was observed in wild-type roots grafted withplants expressing GUS::tRNA^(Ile) (n=0/57 grafts). Again, RT-PCR assaysconfirmed the presence of GUS::tRNA^(Met) and GUS::tRNA^(Gly), and theabsence of GUS::tRNA^(Ile) transcripts in wild-type roots after grafting(FIG. 4D). Notably, the reverse grafts with transgenic roots andwild-type scions indicate that the shoot-to-root mobile GUS::tRNA^(Met)fusion transcript is not moving from root to shoot (n=0/36 grafts) andthat GUS::tRNA^(Gly) barely moves from root to shoot (n=3/26 grafts).

To learn whether the entire tRNA sequence is required or whether asubsequence is sufficient to mediate mRNA mobility, tRNA^(Met) deletionconstructs lacking the assigned dihydrouridine (D)-, anticodon (A)-, orTψC (T)-arm/loop structures and combinations thereof (FIG. 4C) wereused. Again, plants expressing these GUS mRNA fusion constructs weregrafted with wild-type plants and then tested for GUS activity and forfusion transcript presence. As indicated by GUS and RT-PCR assays, theΔD, ΔDT, and ΔDA, but not the ΔAT tRNA^(Met) deletion construct, weresufficient to mediate GUS transport into wild-type roots and, with avery low frequency, to scion leaves (FIGS. 4C-4D). Presence of theGUS::ΔDtRNA^(Met) transcript and translation in phloem-associated cellsof wild-type roots and leaves shows that only part of the tRNA^(Met)sequence is required to trigger mobility. This also demonstrates that A-and TψC hairpin-loop sequences have redundant roles in triggering mRNAtransport as only deletions of both, the A and TψC hairpin-loopsequences, eliminated mobility of the GUS fusion transcript.

In order to elucidate whether tRNA sequences or related TLS motifsconfer mRNA mobility, it was evaluated whether the endogenous mobilemRNA population found in A. thaliana is enriched in tRNA sequencesrelated to viral TLS. For this purpose, the A. thaliana graft-mobiletranscriptome database (n=3606) (Thieme et al., 2015) was screened forpresence of TLS motifs in the mRNA UTRs and coding sequences (CDS) (FIG.7A). Scans were performed for sequence-independent structure motifsusing a provided consensus tRNA descriptor (Macke et al., 2001) whichrecognises tRNA stem-loop arrangements (FIGS. 8A-8C). The analysisrevealed that a significant number of mobile transcripts found in A.thaliana (11.4%; n=411 of 3606) or grapevine (Vitis vinifera) (7.5%;n=249 of 3333) harbour a TLS motif that is found enriched in the CDS and3′UTR (FIG. 7A). Furthermore, annotated tRNA genes were over-representedin close proximity of genes encoding mobile transcripts. Independent ofDNA-strand assignment, 158 of 1,125 genes flanked by a tRNA geneproduced mobile RNAs, and of these 158 cases, 113 are located within1,000 bp distance intervals (FIG. 7B).

Example 3

tRNA-Like-Structure-Mediated mRNA Mobility is Important for NormalDevelopment and Function

Introduction

To confirm the findings from Examples 1 and 2, and to substantiate thenotion that TLSs play a role in transcript mobility of endogenous genes,insertion mutants of the CHOLINE KINASE 1 (CK1; TAIR #AT1G71697) gene,producing a graft mobile transcript (Thieme et al., 2015), which is inclose proximity of a tRNA^(Gly) locus and which produces a bicistronictranscript according to the paired end sequencing data were analysed.

Methods

Grafting

The detailed procedure of Arabidopsis inflorescence stem grafting usedfor CK1 mobility assays was performed as described (Nisar et al., 2012)and samples were harvested for RNA extraction and RT-PCR detection oneweek after grafting.

Quantitative RT-PCR

Quantitative Real-time PCR was performed according to the SYBR Greenmethod in a 5 μl volume using 4 μg total RNA, 2.5 μl SYBR Green MasterMix (Applied Biosystems), 0.2 μM forward and reverse primers. For eachgenomic confirmed ck1 mutant RNA from 3-5 individual plants was isolatedand used. At least three technical replicas were performed. An ABISystem Sequence Detector (Applied Biosystems 7900HT fast Real time PCR)was used with the following regiment of thermal cycling: Stage 1: 1cycle, 2 minutes at 50° C.; Stage 2: 1 cycle, 10 minutes at 95° C.;Stage 3: 40 cycles, 15 seconds at 95° C., 1 minute 60° C. Dissociationstage: 15 seconds at 95° C., 15 seconds at 60° C., 15 seconds at 95° C.Oligonucleotides used for RT-PCR are listed in Table 2.

Results

CK1 catalyses the reaction of choline to phosphatidylcholine (and theCK1 transcript is bicistronic, harbouring a tRNA^(Gly) (TAIR #AT1G71700)sequence in the 3′UTR region (CK1::tRNA^(Gly)). To test whether CK1 mRNAmobility depends on tRNA^(Gly) presence in the 3′UTR, two SALK T-DNAinsertion lines for grafting experiments: ck1.1 (SALK_070759) and ck1.2(SALK_023420) were first confirmed and then used (FIG. 7D). In ck1.1mutants the T-DNA is located within the first intron, while ck1.2mutants have a T-DNA insertion between the stop codon of the CK1 geneand the annotated tRNA^(Gly) sequence, causing the coding sequence ofCK1 and the tRNA^(Gly) sequence to be spaced far apart from each other.

Arabidopsis stem grafting experiments were performed with ck1.2 and wildtype (Col-0) and assayed for the presence of wild-type CK1::tRNA^(Gly)and truncated ck1.2 mRNA in stock and scion samples via RT-PCR (FIG.7E).

Although ck1.2 mutants produce a full-length CK1 poly(A) transcriptcontaining all protein-coding sequences, the truncated transcriptlacking the tRNA^(Gly) sequence could not be detected in wild-typesamples. In contrast, wild-type CK1::tRNA^(Gly) transcript was presentin both ck1.2 scion and ck1.2 stock tissue samples. This suggests thatthe CK1::tRNA^(Gly) transcript was bi-directionally mobile from stock toscion (FIG. 7E), whereas the mutant ck1.2 transcript lacking thetRNA^(Gly) sequence was not transported over graft junctions. Thus, thegraft-mobility of the endogenously produced bicistronic CK1::tRNA^(Gly)transcript depends on the presence of the 3′UTR tRNA^(Gly) sequence.

As lack of detectable ck1.2 transcript mobility could be a result of lowexpression levels, quantitative RT-PCR assays were performed to evaluateCK1 expression levels in the two ck1.1 and ck.2 mutants and in wild-typeplants. Here, only minimal expression could be detected in the ck1.1mutant, whereas CK1 transcript levels in the ck1.2 mutant were similarto that found in the wild type (FIG. 7F). Despite the fact that similarlevels of CK1 poly(A)-RNA transcript were produced by wild-type andck1.2 mutant plants, both the ck1.2 line and the ck1.1 line showed asignificant decrease in rosette leaf size compared to wild type (FIG.7F). This implies that not only CK1 mRNA presence in the expressingcells is required for normal growth behaviour of Arabidopsis, but alsothe mobility of the CK1 mRNA throughout the plant.

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The invention claimed is:
 1. A method for changing the intercellularmobility of an mRNA of a gene in a plant, comprising: modifying a tRNAthat is present in the mRNA by mutating the sequence of the tRNA in thegene from which the mRNA is transcribed to abolish systemic movement ofthe mRNA; or including the sequence of a tRNA in the transcribed part ofthe gene not comprising a tRNA to confer systemic movement to the mRNA,wherein the tRNA comprises an anticodon selected from the groupconsisting of AGC, CGC, UGC, ACG, CCG, CCU, UCG, GUU, GUC, GCA, CUG,UUG, CUC, UUC, CCC, GCC, AAU, AAG, CAA, UAA, UAG, CUU, UUU, CAU, GAA,AGG, CGG, UGG, AGA, GCU, AGU, UGU, CCA, GUA, CAC and UAC, and whereinthe tRNA is an incomplete tRNA that lacks the D stem-loop, the D and Tstem-loops, or the D and A stem-loops.
 2. The method as claimed in claim1, wherein mutating the sequence of the incomplete tRNA comprisesdeleting the sequence of the tRNA from the gene, mutating the sequenceof the tRNA to change the tridimensional configuration thereof, orinserting a genetic element into the gene to remove the tRNA from thetranscribed part of the gene.
 3. The method as claimed in claim 1,wherein mutating the tRNA comprises deleting part of the transcribedsequence thereof, mutating one or more nucleotides involved in theformation of a stem-loop structure in the tRNA, or inserting one or morenucleotides in a stem-loop of the tRNA to change its tridimensionalconfiguration.
 4. The method as claimed in claim 1, wherein includingthe coding sequence of an incomplete tRNA in the transcribed part of thegene is by introducing the complementary sequence of the incomplete tRNAin a DNA construct comprising the complementary sequence of the mRNA. 5.The method as claimed in claim 4, wherein the incomplete tRNA isintroduced in the 3′ untranslated region of the mRNA, or in the 5′untranslated region of the mRNA.
 6. The method as claimed in claim 4,wherein the DNA construct is for stable integration in the genome of theplant, for transient expression in the plant, or for in vitrotranscription.
 7. The method as claimed in claim 1, comprising: a)introducing the complementary sequence of an incomplete tRNA in thetranscribed part of the gene in vitro; b) transcribing the genecomprising the incomplete tRNA in vitro; and c) introducing the mRNAthus obtained into the plant.
 8. The method as claimed in claim 1,wherein the incomplete tRNA is selected from the group consisting oftRNA^(Ala), tRNA^(Arg), tRNA^(Asn), tRNA^(Asp), tRNA^(Cys), tRNA^(Gln),tRNA^(Glu), tRNA^(Gly), tRNA^(His), tRNA^(Leu), tRNA^(Lys), tRNA^(Met),tRNA^(Phe), tRNA^(Pro), tRNA^(Ser), tRNA^(Thr), tRNA^(Trp), tRNA^(Tyr),tRNA^(Val).
 9. The method as claimed in claim 1, wherein the change inmobility results in a loss of the function of the gene.
 10. The methodas claimed in claim 1, wherein the change in mobility results in ectopicproduction of a product of the gene in the plant.
 11. A method forchanging the intercellular mobility of an mRNA of a gene in a plant,wherein said plant consists of a rootstock of a first plant upon which ascion of a second plant has been grafted, and wherein the intercellularmobility of an mRNA of a gene in the first and/or the second plant hasbeen changed according to the method of claim 1.